Amperometric immunosensor

ABSTRACT

An electrochemical immunosensor system with reduced interference, comprising: a first immunosensor that generates an electrochemical signal based on the formation of a sandwich between an immobilized antibody, a target analyte and a labeled antibody, wherein a portion of the signal arises from non-specific binding of the labeled antibody in the region of the first immunosensor, and
         a second immunosensor that acts as an immuno-reference sensor and generates a signal that is the same as or predictably related to the degree of non-specific binding which occurs in the region of the first immunosensor, and has an immunocomplex between an immobilized antibody and an endogenous or exogenous protein that is in the sample and that is not the target analyte.

This application is a divisional of co-pending U.S. application Ser. No.10/658,529, filed Sep. 10, 2003, which is incorporated herein byreference in its entirety.

I. FIELD OF THE INVENTION

An apparatus and method for rapid in situ determination of analytes inliquid samples that is capable of being used in the point-of-careclinical diagnostic field, including use at accident sites, emergencyrooms, in surgery, in intensive care units, and also in non-medicalenvironments.

II. BACKGROUND OF THE INVENTION

The invention relates to an apparatus and its method of use fordetermining the presence or concentrations of analytes in a liquidsample with single-use disposable cartridges adapted for conductingdiverse real-time or near real-time assays of analytes.

In specific embodiments, the invention relates to the determination ofanalytes in biological samples such as blood using electrochemicalimmunosensors or other ligand/ligand receptor-based biosensors. Theinvention further relates to a reference-immunosensor for use with animmunosensor to reduce the effect of interferences in an immunoassay, italso relates to reducing the effect of cellular components, includingleukocytes and erythrocytes, on an immunoassay performed in awhole-blood sample.

A multitude of laboratory tests for analytes of interest are performedon biological samples for diagnosis, screening, disease staging,forensic analysis, pregnancy testing, drug testing, and other reasons.While a few qualitative tests, such as pregnancy tests, have beenreduced to simple kits for the patient's home use, the majority ofquantitative tests still require the expertise of trained technicians ina laboratory setting using sophisticated instruments. Laboratory testingincreases the cost of analysis and delays the results. In manycircumstances, delay can be detrimental to a patient's condition orprognosis, such as for example the analysis of markers indicatingmyocardial infarction. In these critical situations and others, it wouldbe advantageous to be able to perform such analyses at the point ofcare, accurately, inexpensively, and with a minimum of delay.

A disposable sensing device for measuring analytes in a sample of bloodis disclosed by Lauks in U.S. Pat. No. 5,096,669. Other devices aredisclosed by Davis, et al., in U.S. Pat. Nos. 5,628,961 and 5,447,440for a clotting time. These devices employ a reading apparatus and acartridge that fits into the reading apparatus for the purpose ofmeasuring analyte concentrations and viscosity changes in a sample ofblood as a function of time. A potential problem with such disposabledevices is variability of fluid test parameters from cartridge tocartridge due to manufacturing tolerances or machine wear. Zelin, U.S.Pat. No. 5,821,399 discloses methods to overcome this problem usingautomatic flow compensation controlled by a reading apparatus usingconductimetric sensors located within a cartridge. U.S. Pat. Nos.5,096,669, 5,628,961, 5,447,440, and 5,821,399 are hereby incorporatedin their respective entireties by reference.

Antibodies are extensively used in the analysis of biological analytes.For a review of basic principles see Eddowes, Biosensors 3:1-15, 1987.U.S. Pat. No. 5,807,752 to Brizgys discloses a test system in which asolid phase is impregnated with a receptor for an analyte of interest. Asecond analyte-binding partner attached to aspectroscopically-determinable label and a blocking agent is introduced,and the spatial distribution of the label is measured. Spectroscopicmeasurements require a light transducer, typically a photomultiplier,phototransistor, or photodiode, and associated optics that may be bulkyor expensive, and are not required in electrochemical methods, in whichan electrical signal is produced directly.

Electrochemical detection, in which binding of an analyte directly orindirectly causes a change in the activity of an electroactive speciesadjacent to an electrode, has also been applied to immunoassay. For areview of electrochemical immunoassay, see: Laurell, et al., Methods inEnzymology, vol. 73, “Electroimmunoassay”, Academic Press, New York,339, 340, 346-348 (1981).

U.S. Pat. No. 4,997,526 discloses a method for detecting an analyte thatis electroactive. An electrode poised at an appropriate electrochemicalpotential is coated with an antibody to the analyte. When theelectroactive analyte binds to the antibody, a current flows at theelectrode. This approach is restricted in the analytes that can bedetected; only those analytes that have electrochemical midpointpotentials within a range that does not cause the electrode to performnon-specific oxidation or reduction of other species present in thesample by the electrode. The range of analytes that may be determined isextended by the method disclosed in U.S. Pat. No. 4,830,959, which isbased upon enzymatic conversion of a non-mediator to a mediator.Application of the aforementioned invention to sandwich immunoassays,where a second antibody is labeled with an enzyme capable of producingmediator from a suitable substrate, means that the method can be used todetermine electroinactive analytes.

Microfabrication techniques (e.g., photolithography and plasmadeposition) are attractive for construction of multilayered sensorstructures in confined spaces. Methods for microfabrication ofelectrochemical immunosensors, for example on silicon substrates, aredisclosed in U.S. Pat. No. 5,200,051 to Cozzette, et al., which ishereby incorporated in its entirety by reference. These includedispensing methods, methods for attaching biological reagent, e.g.,antibodies, to surfaces including photoformed layers and microparticlelatexes, and methods for performing electrochemical assays.

In an electrochemical immunosensor, the binding of an analyte to itscognate antibody produces a change in the activity of an electroactivespecies at an electrode that is poised at a suitable electrochemicalpotential to cause oxidation or reduction of the electroactive species.There are many arrangements for meeting these conditions. For example,electroactive species may be attached directly to an analyte (seeabove), or the antibody may be covalently attached to an enzyme thateither produces an electroactive species from an electroinactivesubstrate, or destroys an electroactive substrate. See, M. J. Green(1987) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 316:135-142, for areview of electrochemical immunosensors.

The concept of differential amperometric measurement is well known inthe electrochemical art, see for example jointly owned Cozzette, U.S.Pat. No. 5,112,455. In addition, a version of a differentialamperometric sensor combination is disclosed in jointly owned Cozzette,U.S. Pat. No. 5,063,081. However, these and other references are silenton the concept of an immuno-reference sensor coated with antibody to aplasma protein, that is used in conjunction with an immunosensor for ananalyte.

The prior art contains references to immunosensors for detection ofhuman serum albumin using an antibody to human serum albumin forcapture. These include Paek, (U.S. Pat. No. 6,478,938), Berggren (U.S.Pat. No. 6,436,699), Giaever (U.S. Pat. No. 3,853,467), Yamazoe (JP07260782) and Owaku (JP 05273212). These references are silent on theuse of anti-human serum albumin antibody, or other antibodies forestablishing an immuno-reference sensor for use in conjunction with animmunosensor.

The following patents address various means for correcting an analyticaldetermination for the effect of hematocit. U.S. Pat. No. 6,106,778 usessample that is diluted and a Coulter-type cell counter to determine theerythrocyte cell count from which hematocrit is calculated. This is usedto correct the result of an immunoassay. There is no anticipation of theuse of a bulk conductivity sensor and an immunosensor, or making themeasurements in undiluted blood. U.S. Pat. No. 6,475,372 teaches amethod for correcting an analyte concentration for hematocrit based ontwo amperometric measurements at opposite polarities. U.S. Pat. No.4,686,479 provides a sample ion correction for hematocrit measurementsusing the combination of an ion sensor and a conductivity sensor.

U.S. Pat. No. 5,081,063 discloses the use of permselective layers forelectrochemical sensors and the use of film-forming latexes forimmobilization of bioactive molecules, incorporated here by reference.The use of poly(vinyl alcohol) (PVA) in sensor manufacture is describedin U.S. Pat. No. 6,030,827 incorporated here by reference. Vikholm (US2003/0059954A1) teaches antibodies directly attached to a surface with abiomolecule repellant coating, e.g., PVA, the surface in the gapsbetween antibodies, and Johansson (U.S. Pat. No. 5,656,504) teaches asolid phase, e.g., PVA, with antibodies immobilized thereon. U.S. Pat.Nos. 6,030,827 and 6,379,883 teach methods for patterningpoly(vinylalcohol) layers and are incorporated by reference in theirentirety

With regard to amperometric measurements, there are several means knownin the art for reducing the importance of the non-Faradaic component ofthe signal, thus increasing sensitivity. These include newerelectrochemical methods, e.g., using square wave voltammetry in place ofchronoamperometry, and chemical means, e.g., an alkyl thiol reagent topassivate an electrode surface.

Various devices and methods for sealing a biological sample, e.g., bloodinto an analytical system for doing blood tests have been devisedincluding; jointly owned Lauks, U.S. D 337,164; Lauks, U.S. Pat. No.5,096,669; Lauks, U.S. Pat. No. 5,779,650; Lauks, U.S. Pat. No.5,666,967; Lauks, U.S. Pat. No. 5,653,243; Lauks, U.S. Pat. No.5,638,828 and Lauks, U.S. Pat. No. 6,010,463; as well as Cuppoletti,U.S. Pat. No. 5,208,649; Nurse, U.S. Pat. No. 5,254,315; Kilcoin U.S.Pat. No. 6,395,235 and Strand US 2002/0155033. However, these do notdisclose a sealing element that is slidably movable over at least aportion of a planar surface to displace excess fluid away from a sampleentry port, so as to seal a volume of fluid within a holding chamber andinhibit the fluid from prematurely breaking through a capillary stop.

III. SUMMARY OF THE INVENTION

One object of the invention is to provide an electrochemicalimmunosensor system with reduced interference, comprising:

a first immunosensor that generates an electrochemical signal based onthe formation of a sandwich between an immobilized antibody, a targetanalyte and a labeled antibody, wherein a portion of the signal arisesfrom non-specific binding of the labeled antibody in the region of thefirst immunosensor, and

a second immunosensor that acts as an immuno-reference sensor andgenerates a signal that is the same as or predictably related to thedegree of non-specific binding which occurs in the region of the firstimmunosensor, and has an immunocomplex between an immobilized antibodyand an endogenous or exogenous protein that is in the sample and that isnot the target analyte.

Another object is to provide a method for assaying a target analytewhile reducing interference in an electrochemical immunosensor system,comprising:

contacting a sample containing a target analyte plus an endogenous orexogenous protein, which is not the target analyte, with an immunosensorsystem comprising

a first immunosensor that generates an electrochemical signal based onthe formation of a sandwich between an immobilized antibody, a targetanalyte and a labeled antibody, wherein a portion of the signal arisesfrom non-specific binding of the labeled antibody in the region of thefirst immunosensor,

a second immunosensor that acts as an immuno-reference sensor andgenerates a signal that is the same as or predictably related to thedegree of non-specific binding which occurs in the region of the firstimmunosensor, and has an immuno complex between an immobilized antibodyand said endogenous or exogenous protein,

washing said first and second immunosensors with a wash fluid, and

using the signal from the first immunosensor and the signal from thesecond immunosensor to determine a corrected analyte concentration inthe sample.

Another object is to provide an immunoassay device for measuring ananalyte in blood while reducing interference associated with leukocytes,comprising:

providing a conduit for receiving a whole-blood sample in an immunoassaydevice, said conduit containing a salt reagent and means for treatingthe sample sufficient to increase the ionic strength of the sample andthereby reduce interference from the buffy coat when the sample contactsthe immunosensor.

Another object is to provide a method for measuring an analyte in bloodin an immunoassay device while reducing interference associated withleukocytes, comprising:

adding a salt reagent to a whole-blood sample for an immunoassay deviceto increase the ionic strength of the sample and thereby reduceinterference from the buffy coat when the sample contacts theimmunosensor.

Another object is to provide an electrochemical immunosensor system forblood with reduced interference, comprising:

an immunosensor for blood samples that generates an electrochemicalsignal based on the formation of a sandwich between a first immobilizedantibody to a target analyte, the target analyte, and a labeledantibody,

wherein the sensing surface of said immunosensor contains a secondimmobilized antibody covering at least a portion thereof that forms animmunocomplex between an endogenous or exogenous protein that is in thesample and that is not the target analyte.

Another object is to provide an electrochemical immunoassay device formeasuring an analyte in blood and correcting for the hematocrit of thesample to give an equivalent plasma analyte concentration, comprising:

providing a conduit for receiving a blood sample in an electrochemicalimmunoassay device, said conduit containing an immunosensor and a bulkconductivity sensor,

providing computation means for processing signals from said sensors anddetermining the equivalent plasma analyte concentration.

Another object is to provide an amperometric immunosensor, comprising:an electrochemical sensing surface, for a blood sample, having a porouspolyvinylalcohol layer patterned to cover at least a portion of thesurface such that said layer attenuates background current from a bloodsample to at least half the background current obtained in the absenceof the layer.

Another object is to provide a method of performing an electrochemicalimmunoassay for an analyte in blood and correcting for the hematocrit ofthe sample, comprising:

(a) contacting an immunosensor with a blood sample,

(b) contacting said blood sample with a bulk conductivity sensor andmeasuring its resistance,

(c) contacting the immunosensor and conductivity sensor with an aqueoussolution having known conductivity and containing reagents sufficient togenerate a detectable product related to the amount of analyte bound tothe immunosensor,

(d) measuring the signal at the immunosensor generated by said product,

(e) converting by means of an algorithm the signal from the immunosensorto an analyte concentration;

(f) calculating the hematocrit value of the blood sample from themeasured resistance of the blood sample, and

(g) correcting the calculated analyte concentration for the hematocritof the sample.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

These and other objectives, features and advantages of the presentinvention are described in the following detailed description of thespecific embodiments and are illustrated in the following Figures inwhich:

FIG. 1 is an isometric top view of an immunosensor cartridge cover.

FIG. 2 is an isometric bottom view of an immunosensor cartridge cover.

FIG. 3 is a top view of the layout of a tape gasket for an immunosensorcartridge.

FIG. 4 is an isometric top view of an immunosensor cartridge base.

FIG. 5 is a schematic view of the layout of an immunosensor cartridge.

FIG. 6 is a schematic view of the fluid and air paths within animmunosensor cartridge, including sites for amending fluids with dryreagents.

FIG. 7 illustrates the principle of operation of an electrochemicalimmunosensor.

FIG. 8 is a side view of the construction of an electrochemicalimmunosensor with antibody-labeled particles not drawn to scale.

FIG. 9 is a top view of the mask design for the conductimetric andimmunosensor electrodes for an immunosensor cartridge.

FIG. 10 illustrates the electrochemical responses of immunosensorsconstructed with an anti-HCG antibody when presented with 50 mIU/mL ofHCG.

FIG. 11 illustrates the electrochemical response (current versus time)of an immunosensor constructed with an anti-HCG antibody when presentedwith various amounts of HCG from 0 to 50 mIU/mL.

FIG. 12 illustrates the maximum current obtained when an immunosensorconstructed with an anti-HCG antibody is presented with various amountsof HCG.

FIG. 13 is a schematic illustration of enzymatic regeneration of anelectroactive species.

FIG. 14 illustrates segment forming means.

FIG. 15 is a top view of the preferred embodiment of an immunosensorcartridge.

FIG. 16 is a schematic view of the fluidics of the preferred embodimentof an immunosensor cartridge.

FIG. 17 illustrates the electrochemical response (current versus time),and other responses, of a preferred embodiment of an immunosensor.

FIG. 18 illustrates the cartridge device with a slidable sealing elementfor closing the blood entry port in the closed position.

FIG. 19 illustrates the cartridge device with a slidable sealing elementfor closing the blood entry port in the open position.

FIG. 20 illustrates a perspective view of the slidable sealing element.

FIG. 21 illustrates a side view of the slidable sealing element.

FIG. 22 illustrates the decreased background current at a troponin Iimmunosensor as a function of sodium chloride added to the sample.Addition of about 100 mM sodium ion brings the sample concentration toabout 240 mM (assuming a typical blood sample sodium ion concentrationof about 140 mM).

FIG. 23 is a schematic representation of a whole blood immunoassay.

FIG. 24 is a schematic representation of a whole blood immunoassay withan immunoreference electrode.

V. DETAILED DESCRIPTION OF THE INVENTION

The present invention permits rapid in situ determinations of analytesusing a cartridge having an array of analyte sensors and means forsequentially presenting a sample and a fluid (amended or not) to theanalyte array. The cartridges are designed to be preferably operatedwith a reading device, such as that disclosed in U.S. Pat. No. 5,096,669to Lauks, et al., issued Mar. 17, 1992, or U.S. Pat. No. 5,821,399 toZelin, issued Oct. 13, 1998, which are both hereby incorporated byreference in their respective entireties.

The invention provides cartridges and methods of their use forprocessing liquid samples to determine the presence or amount of ananalyte in the sample. The cartridges contain a metering means, whichpermits an unmetered volume of sample to be introduced, from which ametered amount is processed by the cartridge and its associated readingapparatus. Thus the physician or operator is relieved of accuratelymeasuring the volume of the sample prior to measurement saving time,effort, and increasing the accuracy and reproducibility.

The metering means comprises an elongated sample chamber bounded by acapillary stop and having along its length an air entry point. Airpressure exerted at the air entry point drives a metered volume of thesample past the capillary stop. The metered volume is predetermined bythe volume of the sample chamber between the air entry point and thecapillary stop.

Slidable Closure

The cartridge may have a closure means for sealing the sample port in anair-tight manner. This closure device is slidable with respect to thebody of the cartridge and provides a shearing action that displaces anyexcess sample located in the region of the port; reliably sealing aportion of the sample in the holding chamber between the entry port andthe capillary stop. The cartridge is sealed by slidably moving a sealingelement over the surface of the cartridge in a manner that displacesexcess fluid sample away from the sample orifice, seals a volume of thefluid sample within the internal fluid sample holding chamber, andinhibits fluid sample from prematurely breaking through the internalcapillary stop.

The seal obtained by this slidable closure means is irreversible andprevents excess blood from being trapped in the cartridge because theclosure means moves in the plane of the orifice through which bloodenters the cartridge and provides a shearing action that seals bloodbelow the plane of the entry port; moving excess blood, i.e., bloodabove the plane of the orifice, away from the entry port and optionallyto a waste chamber.

Thus, an alternative to the blood entry port closure means comprisingintegrated elements 2, 3, 4 and 9 of cover 1 in FIG. 1 is shown as aseparate slidable element 200 in FIGS. 18, 19, 20 and 21. FIG. 18 showsa cartridge device comprising a modified version of the cover of FIG. 1attached to the base of FIG. 4 with the intervening adhesive layer 21shown in FIG. 3 along with the separate slidable closure element 200. Itis shown in the closed position where it seals the blood entry port inan air-tight manner. FIG. 19 shows the same components as FIG. 18, butwith the slidable closure element in the open position, where the bloodentry port 4 can receive blood. In operation, element 200 is manuallyactuated from the open to the closed position after blood has been addedto the entry port and it enters the holding chamber 34. Any excess bloodin the region of the entry port is moved into an overflow chamber 201 oran adjacent retaining region. This chamber or region may include ablood-absorbing pad or material to retain the excess blood.

The sealing element 200, also shown in FIGS. 20 and 21, has a proximalend and a distal end; the proximal end has at least one anterior prong202 and at least one posterior prong 203, preferably the anterior pronghas a greater thickness relative to the posterior prong when viewing alongitudinal cross section of the sealing element. The prongs areseparated by a gap 204, which permits the slidable movement of saidsealing element relative the cartridge. In operation, when the closureelement is manually actuated the anterior prong slides over and acrossat least a portion of the device's substantially planar surface 205 inthe region of the blood entry port; and the posterior prong slides undera portion of a face 206 of the cartridge opposite its substantiallyplanar surface 205. Over and under are relative terms with respect tothe cartridge. The sealing element may also include a dome-like shape207 on the anterior prong while the posterior prong includes asubstantially planar shape 208 when viewing the longitudinal crosssection of said sealing element, as in FIG. 21. The gap 204 separatingthe prongs runs approximately half the length of said sealing element200.

In operation, the anterior prong remains substantially rigid as thisprong slides over and across the device at 205, while the posteriorprong may flex as this prong slides under 205. The anterior prong mayalso be longer than the posterior prong so that a tip of the anteriorprong extends beyond the tip of the posterior prong. The sealing elementcan also include, at the distal end, anterior prong 209 and posteriorprong 210 separated by a gap 211 that runs approximately a third of thelength of said sealing element. Optionally 209 may includes a fin-likeshape, while 210 may have a substantially planar shape when viewing alongitudinal cross section of the sealing element. Preferably 209 has atip that extends beyond 210. The sealing element is preferably made froma plastic material with mechanical properties and dimensions that permitthe desired degree of flexing. Such materials include polyesters, ABS,acetal polymers and the like, that are suitable for injection molding.

The sealing element also can include a locking feature 212, whichengages after the sealing element covers the blood entry port into agroove 213 in the base of the device. This ensures that the sealingelement remains in the closed position throughout the assay procedure.Engagement of the sealing element in the closed position abuts it in anairtight manner to the region surrounding the blood entry port.Additionally, grooves and proud features may be molded into the sealingelement and cartridge base to assure that when the sealing element ismoved from the open to closed position, it tracks to the desired closedposition completely covering the blood entry port. An additional lockingfeature may be included on prong 203 and the cartridge base.

The blood entry port 4 may be an orifice that is circular, as shown inFIG. 19, or oval and the diameter of the orifice is generally in therange 0.2-5 mm, preferably 1-2 mm, or having a perimeter of 1-15 mm foran oval. The region around the orifice may be selected to be hydrophobicor hydrophilic to control the drop-shape of the applied blood sample topromote entry into the entry port. Optionally, it may be a portion of anadhesive tape material 21 that is capable of forming an airtight sealwith the sealing means.

One advantage of this sealing element is that it prevents blood beingpushed beyond the capillary stop element 25 at the end of the bloodholding chamber 34. The presence of a small amount of blood beyond thecapillary stop is not significant for tests that measure bulkconcentration of an analyte and thus do not depend on sample volume.However, for immunoassay applications where metering of the sample isgenerally advantageous the sealing element improves metering accuracy ofthe device and assures the assayed segment of sample is appropriatelypositioned with respect to the immunosensor, when the analyzer actuatesthe sample within the cartridge's conduits.

In operation, when blood is added to the cartridge it moves to thecapillary stop. Thus sufficient blood for the assay is present when theregion from the capillary stop to the blood entry port, i.e., theholding chamber, contains blood. During the process of filling theholding chamber some blood may remain above the plane of the orifice ofthe entry port. When the sealing element is moved from the opened toclosed position, any blood that is above the entry port is sheared awaywithout trapping additional blood in the act of closure, thus ensuringthat blood does not move beyond 25. In a preferred embodiment, sealingelement 200 is positioned within a few thousandths of an inch above thesurface of the tape gasket 21 of FIG. 3. The entry port is sealed by thesubsequent lowering of the surface of 200 to the adhesive tape gasketwhen it engages locking features 212 and 213. Once this seal is achievedit is essentially irreversible. Furthermore, since the tape isessentially incompressible and the orifice has a small diameter, anyinadvertent pressure applied to the sealing element by the user will notcause the blood to move beyond the capillary stop.

While sealing element 200 and its attendant features are particularlyadvantageous for an immunoassay and DNA testing cartridges, they canalso be used with cartridges that have sensors for other tests includingsodium ion, glucose, activated clotting time and the like. It can beconsidered applicable to any cartridge with an immunosensor,electrochemical sensor, acoustic-wave sensor, optical sensor and thelike.

Bubble-Free Blood Entry into Holding Chamber

A reliable means for introducing more than one drop of blood into theblood holding chamber without entraining bubbles has been developed. Incertain cartridge embodiments that use several drops of blood, it isdesirable that no bubbles form in the holding chamber as this can affectthe assay. For example, in a coagulation assay, e.g., prothrombin time(PT), the cartridge needs to work with a few drops of blood from afingerstick.

The blood-entry port can be designed to receive multiple drops of bloodwithout successive drops causing trapped bubbles to form in the holdingchamber 34 by treating the holding chamber with a Corona and/or areagent cocktail. Surface 34 is first Corona treated to provide chargedsurface groups that will promote spreading of the aqueous printedcocktail.

The use of corona treatments on disposable medical devices is well knownin the art. It is an effective way to increase the surface activity ofvirtually any material, e.g., plastics such as polyethylene,polypropylene, nylon, vinyl, PVC, and PET; metallized surfaces, foils,paper, and paperboard stock. This treatment makes them more receptive toinks, coatings, and adhesives. In practice the material being treated isexposed to an electrical discharge, or “corona.” Oxygen molecules in thedischarge area break into atoms and bond to molecules in the materialbeing treated, resulting in a chemically activated surface. Suitableequipment for corona treatments is commercially available (e.g., CorotecCorp., Farmington, Conn.). The process variables include the amount ofpower required to treat the material, the material speed, the width, thenumber of sides to be treated, and the responsiveness of a particularmaterial to corona treatment, which variables can be determined by askilled operator. The typical place to install a corona treatment isin-line with the printing, coating, or laminating process. Anothercommon installation is directly on a blown film or cast film extrudersince fresh material is more receptive to corona treatment.

In general the cocktail may contain a water-soluble protein, an aminoacid, a polyether, a polymer containing hydroxyl groups, a sugar orcarbohydrate, a salt and optionally a dye molecule. One or more of eachcomponent can be used. In one embodiment the cocktail contains bovineserum albumin (BSA), glycine, methoxypolyethylene glycol, sucrose andoptionally bromophenol blue to provide color that aids visualizing theprinting process. A salt would be a component for an immunoassay.Typically, 1-20 uL of cocktail is printed onto the holding chamberbefore being assembled with its cover and allowed to air dry.

A preferred composition is given in Example 6 for a coagulation assay,in which the amount of each component printed in the sample holdingchamber in the base (coating) is shown. The components are BSA, glycine,methoxypolyethylene glycol, sucrose and bromophenol blue. The sampleholding chamber in the cartridge base is corona treated prior toprinting. The cartridge cover need not be treated with either corona orcocktail although it may be advantageous for some assays. In a preferredembodiment there is no special treatment for the cover and no treatmentaround the blood entry orifice.

Printing is automated and based on a microdispensing system, including acamera and computer system to align components, as disclosed in U.S.Pat. No. 5,554,339, where the wafer chuck is replaced by a means forfeeding the plastic cartridge bases to the dispensing head.

In operation, for efficient draw of blood into the holding chamber ofsmall volumes (about 20 uL and less) it is desirable to have a highcapillarity to clear the entry port of a first drop of blood so that asecond can be added without spilling around the port due the first onebeing still partially there. The results for the corona (C) and reagent(R) treatment combinations are as follows:

1. C no, R no: No reliable blood draw is observed.2. C no, R yes: Reagent does not coat holding chamber reliably. As for1.3. C yes, R no: Rapid blood draw is good for several weeks but candegrade with time.4. C yes, R yes: Rapid blood draw is good and lasts for +6 months (atypical target for product shelf-life).

The reagent concentration must be low so that it works as desired, butdoes not interfere with the assay, e.g., components in blood that giverise to the coagulation cascade, as in PT, APTT and ACT assays. Oneskilled in the art will not need unreasonable experimentation here,i.e., freshly made cartridges with processes 3 and 4 should have thesame assay results. The actual concentrations will depend on the designof the cartridge, dimensions, plastics etc.

Electrochemical Immunosensor with Reduced Background

The signal-to-noise ratio is a well known factor in any measurement.Here, we provide a means for reducing the noise (or background signal)in an amperometric immunoassay. It has been discovered that theimmunosensor exhibits reduced non-Faradaic (background or chargingcurrent) signal, by adding certain porous layers interposed between theelectrode and the antibody layer. This type of assay relies on measuringcurrents in the nanoampere range with comparatively low concentrationsof electroactive species (e.g., p-aminophenol), thus the backgroundcurrent can be a significant portion of the measured signal.

It has been discovered that an intervening polyvinyl alcohol (PVA) layerof about 0.5-5.0 micron thickness (preferably 0.6-1.0 micron) placedbetween the electrode and the antibody reagent layer significantlyattenuates the background component. An advantage of PVA as thebackground-reducing layer is that noise is reduced without appreciablyaffecting the Faradaic component of the signal. While the PVA layerreduces the diffusion coefficient of small molecules by about 50% it hasbeen found that it does not change the current at the coated electrodes,for two reasons. First, with PVA layers of about 1 micron thickness, thedetected electroactive species is present in a diffusion layer of atleast ten times that thickness, so there is little decrease in transportdue to the PVA layer. Second, a steady-state current is measured in theimmunosensor which is effectively independent of the transport rate andelectrode kinetics, but is a function of the enzymatic rate ofproduction of the detectable species, such as p-aminophenol generatedfrom p-aminophenol phosphate by the enzyme alkaline phosphatase(attached to the second antibody).

Wafer-level microfabrication of a preferred embodiment of theimmunosensor is as follows. The base electrode (94 of FIG. 9) consistsof a square array of 7 um gold disks on 15 um centers. The array coversa circular region approximately 600 um in diameter, and is achieved byphoto-patterning a thin layer of polyimide of thickness 0.35 um over asubstrate made from a series of layers comprising Si/SiO2/TiW/Au. Thearray of 7 um microelectrodes affords high collection efficiency ofelectroactive species with a reduced contribution from anyelectrochemical background current associated with the capacitance ofthe exposed metal. The inclusion of a PVA layer over the metalsignificantly enhances the reduction of background currents.

The porous PVA layer is prepared by spin-coating an aqueous mixture ofPVA plus a stilbizonium photoactive, cross-linking agent over themicroelectrodes on the wafer. The spin-coating mixture optionallyincludes bovine serum albumin (BSA). It is then photo-patterned to coveronly the region above and around the arrays and preferably has athickness of about 0.6 um.

The improved background screening properties of the PVA layer wereestablished by including alkaline phosphatase (ALP) into the patternedlayer to assess collection efficiency and by comparing the backgroundand p-aminophenol currents in solutions containing ALP. The PVA layerwas associated with a reduction in background current of about a factorof three, without any significant attenuation of the p-aminophenolsignal.

Without being bound to theory, suppression of the background current islikely to involve a degree of permselectivity towards p-aminophenol overelectrochemical contaminants and other species that adsorb at theelectrode surface which modify the double layer capacitance.Alternatively, the layer may have an effect on the electrode surfacethat preferentially reduces the rate of electrochemically irreversible(background) reactions, while affecting relatively reversible reactions,e.g., p-aminophenol, to a lesser degree. Also, the absorbent nature ofthe PVA layer may aid in maintaining continuity (conductivity) during anamperometric analysis. Failure to maintain conductivity may result in adrifting potential that would contribute to background noise.

Immuno-Reference Sensor

The general concept of differential measurement is known in theelectrochemical and sensing arts. A novel means for reducing interferingsignals in an electrochemical immunosensing systems is now described.However, while it is described for pairs of amperometric electrochemicalsensors it is of equal utility in other electrochemical sensing systemsincluding potentiometric sensors, field effect transistor sensors andconductimetric sensors. It is also applicable to optical sensors, e.g.,evanescent wave sensors and optical wave guides, and also other types ofsensing including acoustic wave and thermometric sensing and the like.Ideally, the signal from an immunosensor (IS) is derived solely from theformation of a sandwich between an immobilized antibody (the analyte)and a second antibody that is labeled, wherein the label (e.g., anenzyme) reacts with a substrate to form a detectable product (1).

Surface-Ab1˜analyte˜Ab2-enzyme enzyme+S→P  (1)

It is known that some of the second antibody can bind non-specificallyto the surface (2, 3) and might not be washed away completely from theregion of the immunosensor (up to approx. 100 microns away) during thewashing step, giving rise to a portion of the total detected productthat is not a function of the surface-Ab1˜analyte binding reacting; thatis, an interfering signal.

Surface˜Ab2-enzyme enzyme+S→P  (2)

Surface˜analyte-Ab2-enzyme enzyme+S→P  (3)

A second immunosensor can be placed in the cartridge that acts as animmuno-reference sensor (IRS) and gives the same (or a predictablyrelated) degree of non-specific binding as occurs on the primaryimmunosensor. Interference can be reduced by subtracting the signal ofthis immuno-reference sensor from that of the immunosensor, i.e., thenon-specific binding component of the signal is removed, improving theperformance of the assay (4).

Corrected signal=IS−IRS  (4)

The immuno-reference sensor is preferably the same in all significantrespects (e.g., dimensions, porous screening layer, latex particlecoating, and metal electrode composition) as the immunosensor exceptthat the capture antibody for the analyte (for instance, cTnI) isreplaced by an antibody to a plasma protein that naturally occurs insamples (both normal and pathological) at a high concentration. Theimmunosensor and reference immunosensor may be fabricated as adjacentstructures 94 and 96, respectively, on a silicon chip. While thepreferred embodiment is described for a troponin I assay, this structureis also useful for other cardiac marker assays including troponin T,creatine kinase MB, procalcitonin, BNP, proBNP, myoglobin and the like,plus other sandwich assays used in clinical diagnostics.

Examples of suitable antibodies that bind to plasma proteins includeantibodies to human serum albumin, fibrinogen and IgG fc region, withalbumin being preferred. However, any native protein or blood componentthat occurs at a concentration of greater than about 100 ng/mL can beused if an appropriate antibody is available. The main requirement ofthe protein is being present in sufficient amounts to coat the sensorquickly compared to the time needed to perform the analyte assay. In apreferred embodiment, the protein is present in a blood sample at aconcentration sufficient to bind more than 50% of the available antibodyon the reference immunosensor within about 100 seconds of contacting ablood sample. In general the second immobilized antibody has an affinityconstant of about 1×10(−7) to about 1×10(−15)M. For example, an antibodyto albumin having an affinity constant of about 1×10(−10) M ispreferred, due to the high molar concentration of albumin in bloodsamples, which is about 1×10(−4) M.

It has been found that providing a surface that is covered by nativealbumin derived from the sample significantly reduces the binding ofother proteins and cellular materials which may be present. This methodis generally superior to prior art immunoassays that use conventionalblocking agents to minimize non-specific binding. Because these agentsmust typically be dried down and remain stable for months or yearsbefore use, during which time they may degrade, creating a stickiersurface than desired and resulting in non-specific binding that riseswith age. In contrast the method described here provides a fresh surfaceat the time of use.

An immunosensor for cardiac troponin I (cTnI) with areference-immunosensor for performing differential measurement to reducethe effect of non-specific binding is described next.Carboxylate-modified latex microparticles (supplied by BangsLaboratories Inc. or Seradyn Microparticles Inc.) coated with anti-cTnIand anti-HSA are both prepared by the same method. The particles arefirst buffer exchanged by centrifugation, followed by addition of theantibody, which is allowed to passively adsorb onto the particles. Thecarboxyl groups on the particles are then activated with EDAC in MESbuffer at pH 6.2, to form amide bonds to the antibodies. Any beadaggregates are removed by centrifugation and the finished beads arestored frozen.

It was found that for the anti-human serum albumin (HSA) antibody,saturation coverage of the latex beads results in about a 7% increase inbead mass. Coated beads were prepared using covalent attachment from amixture comprising 7 mg of anti-HSA and 100 mg of beads. Using thispreparation a droplet of about 0.4 mL, comprising about 1% solids indeionized water, was microdispensed (using the method and apparatus ofU.S. Pat. No. 5,554,339, incorporated here by reference) onto aphoto-patterned porous polyvinyl alcohol permselective layer coveringsensor 96, and allowed to dry. The dried particles adhered to the porouslayer and substantially prevented their dissolution in the blood sampleor the washing fluid.

For the troponin antibody, saturation coverage of the latex bead surfaceresulted in a mass increase in the beads of about 10%. Thus by adding 10mg of anti-TnI to 100 mg of beads along with the coupling reagent,saturation coverage was achieved. These beads were then microdispensedonto sensor 94.

In an another embodiment, immunosensor 94 is coated with beads havingboth a plasma protein antibody, e.g., anti-HSA, and the analyteantibody, e.g., anti-cTnI. Latex beads made with the about 2 mg or lessof anti-HSA per 100 mg of beads and then saturation-coated withanti-cTnI provide superior non-specific binding properties at theimmunosensor. It has been found that the slope (signal versus analyteconcentration) of the troponin assay is not materially affected becausethere is sufficient anti-cTnI on the bead to capture the availableanalyte (antigen). By determining the bead saturation concentration fordifferent antibodies, and the slope of an immunosensor having beads withonly the antibody to the target analyte, appropriate ratios of antibodycombinations can be determined for beads having antibodies to both agiven analyte and a plasma protein.

An important aspect of immunosensors having a reference immunosensor isthe “humanizing” of the surface created by a layer of plasma protein,preferably the HSA/anti-HSA combination. This appears to make the beadsless prone to non-specific binding of the antibody-enzyme conjugate. Italso seems to reduce bead variability. Without being bound by theory, itappears that as the sensors are covered by the sample they are rapidlycoated with native albumin due to the anti-HSA surface. This givessuperior results compared to conventional blocking materials which aredried down in manufacturing and re-hydrated; typically after a longperiod in storage. Another advantage to “humanizing” the sensor surfaceis that it provides an extra mode of resistance to human anti-mouseantibodies (HAMA) and other heterophile antibody interferences. Theeffects of HAMA on immunoassays are well known.

Another use of the immuno-reference sensor of the invention is tomonitor the wash efficiency obtained during the analytical cycle. Asstated above, one source of background noise is the small amount ofenzyme conjugate still in solution, or non-specifically absorbed on thesensor and not removed by the washing step. This aspect of the inventionrelates to performing an efficient washing step using a small volume ofwashing fluid, by introducing air segments as mentioned in Example 2.

In operation of the preferred embodiment, which is an amperometricelectrochemical system, the currents associated with oxidation ofp-aminophenol at immunosensor 94 and immuno-reference sensor 96 arisingfrom the activity of ALP, are recorded by the analyzer. The potentialsat the immunosensor and immuno-reference sensor are poised at the samevalue with respect to a silver-silver chloride reference electrode. Toremove the effect of interference, the analyzer subtracts the signal ofthe immuno-reference sensor from that of the immunosensor according toequation (4). Where there is a characteristic constant offset betweenthe two sensors, this also is subtracted. It will be recognized that itis not necessary for the immuno-reference sensor to have all the samenon-specific properties as the immunosensor, only that it beconsistently proportional in both the wash and non-specific bindingparts of the assay. An algorithm embedded in the analyzer can accountfor any other essentially constant, difference between the two sensors.

Use of a differential combination of immunosensor and immuno-referencesensor, rather than an immunosensor alone, provides the followingimprovement to the assay. In a preferred embodiment the cartridge designprovides dry reagent that yields about 4-5 billion enzyme conjugatemolecules dissolved into about a 10 uL blood sample. At the end of thebinding and wash steps the number of enzyme molecules at the sensor isabout 70,000. In experiments with the preferred embodiment there were,on average, about 200,000 (+/−about 150,000) enzyme molecules on theimmunosensor and the reference immunosensor as non-specifically boundbackground. Using a differential measurement with the immuno-referencesensor, about 65% of the uncertainty was removed, significantlyimproving the performance of the assay. While other embodiments may haveother degrees of improvement, the basis for the overall improvement inassay performance remains.

An additional use of this immuno-reference sensor is to detect anomaloussample conditions, such as improperly anti-coagulated samples whichdeposit material throughout the conduits and cause increased currents tobe measured at both the immunosensor and the immuno-reference sensor.This effect is associated with both non-specifically adsorbed enzyme andenzyme remaining in the thin layer of wash fluid over the sensor duringthe measurement step.

Another use of the immuno-reference sensor is to correct signals forwashing efficiency. In certain embodiments the level of signal at animmunosensor depends on the extent of washing. For example, longerwashing with more fluid/air segment transitions can give a lower signallevel due to a portion of the specifically bound conjugate being washedaway. While this may be a relatively small effect, e.g., less than 5%,correction can improve the overall performance of the assay. Correctionmay be achieved based on the relative signals at the sensors, or inconjunction with a conductivity sensor located in the conduit adjacentto the sensors, acting as a sensor for detecting and counting the numberof air segment/fluid transitions. This provides the input for analgorithmic correction means embedded in the analyzer.

In another embodiment of the reference immunosensor with an endogenousprotein, e.g., HSA, it is possible to achieve the same goal by having animmuno-reference sensor coated with antibody to an exogenous protein,e.g., bovine serum albumin (BSA). In this case the step of dissolving aportion of the BSA in the sample, provided as an additional reagent,prior to contacting the sensors is needed. This dissolution step can bedone with BSA as a dry reagent in the sample holding chamber of thecartridge, or in an external collection device, e.g., a BSA-coatedsyringe. This approach offers certain advantages, for example theprotein may be selected for surface charge, specific surface groups,degree of glycosylation and the like. These properties may notnecessarily be present in the available selection of endogenousproteins.

Immunosensor with Improved Precision

An electrochemical immunosensor of the type described here can exhibit abias between whole-blood versus plasma. Historically, immunoassays formarkers such as troponin and the like are measured and reported asplasma or serum values. When these immunosensors are used for analysisof whole-blood, either a correction factor or a means for eliminatingthe bias needs to be employed. It has been found that two aspects ofthis bias can be eliminated: (i) the bias in whole-blood electrochemicalimmunoassays associated with components of the buffy coat (whichconsists of white blood cells and platelets), and (ii) the biasassociated with hematocrit variations between samples.

The buffy coat is a layer of leukocytes and platelets that forms abovethe erythrocytes when blood is centrifuged. It has been observed that awhite cell (or leukocyte) interference occurs on immunosensors havingbeads coated with an analyte antibody, e.g., troponin antibody. Controlexperiments showed that this positive bias is absent in plasma samplesand in blood samples where the buffy coat has been removed. Withoutbeing bound by theory, it appears that leukocytes are able to stick tothe immunosensor and promote non-specific binding of the enzyme-labeledantibodies, which remain bound even after a washing step. It has beenfound that this bias can be partially eliminated by adding a smallamount of an antibody to human serum albumin during bead preparation.When a sample contacts the modified beads, albumin from the samplerapidly coats the beads as described above. Once they are coated with alayer of native albumin the leukocytes do not recognize the beads as anopsonized surface, resulting in the observed effect of limiting theleukocytes' ability to cause the bias.

Another solution to the leukocyte interference problem has also beendiscovered. This bias can be eliminated by increasing the saltconcentration of the blood sample from a normal sodium ion concentrationof about 140 mM to above about 200 mM, preferably to about 230 mM. Forconvenience, the salt's effective concentration is expressed as thesodium ion concentration. FIG. 22 is a graph of the difference in netcurrent at the immunosensor versus added NaCl concentration. In apreferred embodiment, sufficient salt is added to the blood-holdingchamber of the cartridge in a dried down form able to dissolve in thesample prior to the measurement step. Because the holding chamber has avolume of about 10 uL in the preferred cartridge design, this requiresabout 60 ug of NaCl. Without being bound by theory, a mechanism thataccounts for reduced interference may be that the salt causes osmoticshrinkage of the leukocytes, since this is an established phenomenonwith erythrocytes. This interpretation is consistent with theleukocytes' impaired ability to interact with the immunosensor.

Suitable salts are not limited to NaCl, for example KCl, MgCl2, CaCl2,LiCl, NaNO3, Na2SO4 can be used, as well as common buffer salts, e.g.,Tris, MES, phosphate and HEPES. The type of salt and effectiveconcentration range to obviate the buffy coat interference effect can bedetermined by routine experimentation. Other alternatives that have asimilar effect include sugars, DEAE dextran and lactitol. Thesematerials can also be used as the matrix for printing the salt into thecartridge.

In a preferred embodiment salt is added to the sample by coating thewall of the sample holding chamber with a mixture of NaCl, lactitol andDEAE dextran at pH 7.4 using Tris at about 5% total solids. Gelatin,cellulose and PVA can also be used as the support matrix, but thedissolution rate is not quite as fast as the lactitol and DEAE mixture.

It has been found that the addition of salt before the assay can be usedadvantageously in combination with the HSA-antibody coated beads on theimmunosensor.

In addition to salts, other reagents can improve whole-blood precisionin an immunoassay. These reagents should be presented to the bloodsample in a way that promotes rapid dissolution. Support matricesincluding cellulose, polyvinyl alcohol and gelatin (or mixtures thereof)that are coated on to the wall of the blood-holding chamber (or anotherconduit) promote rapid dissolution, e.g., greater than 90% complete inless than 15 seconds.

Other optional additives may be included into the cartridge or used inconjunction with the assay. The anticoagulant heparin can be added toimprove performance in cases where the sample was not collected in aheparinized tube or was not properly mixed in a heparinized tube. Enoughheparin is added so that fresh unheparinized blood will remainuncoagulated during the assay cycle of the cartridge, typically in therange of 2-20 minutes. Goat and mouse IgG can by added to combatheterophile antibody problems well known in the immunoassay art.Proclin, DEAE dextran, Tris buffer and lactitol can be added as reagentstabilizers. Tween 20 can be added to reduce binding of proteins to theplastic, which is the preferred material for the cartridge. It alsoallows the reagents to coat the plastic surface more evenly and acts asan impurity that minimizes the crystallization of sugars, such aslactitol, so that they remain a glass. Sodium azide may be added toinhibit bacterial growth.

Immunosensor with Reduced Hematocrit Interference

Sample hematocrit values may vary widely between immunoassays performedon whole-blood and this can affect the results. A way to eliminate thiseffect has been discovered. Experiments showed that when the bloodsample is placed over the immunosensor in the cartridge, the reagentsthat form the immuno-complex dissolve into the plasma fraction only, notinto the cells, which are predominantly erythrocytes. Erythrocytestypically occupy about 40% of a blood sample, though this can varywidely between patients. The percentage of the blood sample volumeoccupied by these cells is called the hematocrit value. For a givenvolume of blood, the higher the hematocrit value the less plasma volumeis available for a given amount of reagent to dissolve in; thus, theeffective reagent concentration is higher. Therefore, it was observedthat the signal generated in the assay increases with increasinghematocrit. This effect can be corrected for by measuring the hematocritof the sample during the assay. The analyte concentration can then bereported so as to agree with typical laboratory values obtained fromspun samples, i.e., serum or plasma samples where hematocrit equalszero.

We have found that measurement of the bulk conductivity of the samplewith (or without) the dissolved reagents gives an adequate estimate ofthe hematocrit. It is known that the hematocrit is an inverse functionof conductivity, assuming a normal concentration of current-carryingions in the sample. In one embodiment standard curves are created usingsamples with independently determined analyte concentrations andhematocrit values. One skilled in the art will understand that analgorithm can be developed and embedded into the analyzer and used forreal samples, whereby the conductivity measured at an adjacent sensor inthe cartridge is used to estimate hematocrit and correct the signal fromthe immunosensor. For example, the algorithm may simply subtract apercentage of the signal per hematocrit unit in order to correct theresult to a hematocrit of zero, i.e., plasma.

Immunosensor Correction for Buffy Coat and Hematocrit

The embodiments described above provide means for the individualelimination of the buffy coat interference and variable hematocritvalues in electrochemical immunoassays performed on whole-blood samples.It is however desirable to deal with both interferences in the samesample and at the same sensor. The following method assures that theaddition of salt to the sample to eliminate the buffy coat interferencedoes not affect the hematocrit measurement, which is based on aconductivity measurement. Those skilled in the art will recognize thatadding salt to a blood sample with an otherwise normal concentration ofions will increase its conductivity, thus giving an inaccurately lowvalue of hematocrit. The present embodiment minimizes this problem. Ithas been found that when adding salt to the blood holding chamber, theplot of signal versus hematocrit is non-linear because the final plasmavolume and resulting conjugate concentration depend on both the initialhematocrit value and the amount of erythrocyte shrinkage resulting fromadding a fixed amount of salt. It has been found that a plot of signalversus hematocrit provides a parabolic curve if the data are normalizedto plasma, i.e., if signal in plasma is unity, then the curve is thesame regardless of the analyte concentration, e.g., [cTnI]. This is alsotrue if the signal is plotted versus sample conductivity.

The most facile means for correcting data arising from an assay using asalt print is as follows; (i) measure the net signal current for theassay (corrected for the reference immunosensor current) using themethod described above; equation (4), (ii) measure the sampleconductivity after dissolution of the holding chamber salt print, i.e.,make the measurement of conductivity during capture/mixing, (iii) fromthe measured sample conductivity of step 2, calculate the value of thePlasma Normalization Current Function (PNCF)=c1*Cond̂2+c2*Cond+c3(PNCF=unity for plasma), and (iv) divide the net current from step 1, bythe correction factor calculated in step 3 to yield the “plasma[analyte]”.

Regarding the parabolic curve, it has been found that as hematocritincreases from zero to about 30 percent, the PNCF increases from 1.0 toabout 1.3 and as the hematocrit further increases from about 30 to about60, the PNCF decreases back to a value of about 1.0. The advantage ofusing this method is that the algorithm for the correction no longerinvolves discrete estimation of the hematocrit of the sample. Here, thesalt addition can be generalized to increased ionic strength orosmolarity in minimizing the buffy coat interference.

While this is the preferred method for the preferred immunosensor andcartridge elements described above, those skilled in the art willrecognize that other component combinations, particularly for otheranalytes may require re-optimization of the PNCF algorithm usingdifferent constants and the like.

A cartridge of the present invention has the advantage that the sampleand a second fluid can contact the sensor array at different timesduring an assay sequence. The sample and second fluid may also beindependently amended with other reagents or compounds present initiallyas dry coatings within the respective conduits. Controlled motion of theliquids within the cartridge further permits more than one substance tobe amended into each liquid whenever the sample or fluid is moved to anew region of the conduit. In this way, provision is made for multipleamendments to each fluid, greatly extending the complexity of automatedassays that can be performed, and therefore enhancing the utility of thepresent invention.

In a disposable cartridge, the amount of liquid contained is preferablykept small to minimize cost and size. Therefore, in the presentinvention, segments within the conduits are also used to assist incleaning and rinsing the conduits by passing the air-liquid interface ofa segment over the sensor array or other region to be rinsed at leastonce. It has been found that more efficient rinsing, using less fluid,is achieved by this method compared to continuous rinsing by a largervolume of fluid.

Restrictions within the conduits serve several purposes in the presentinvention. A capillary stop located between the sample chamber and firstconduit is used to prevent displacement of the sample in the holdingchamber until sufficient pressure is applied to overcome the resistanceof the capillary stop. A restriction within the second conduit is usedto divert wash fluid along an alternative pathway towards the wastechamber when the fluid reaches the constriction. Small holes in thegasket, together with a hydrophobic coating, are provided to preventflow from the first conduit to the second conduit until sufficientpressure is applied. Features that control the flow of liquids withinand between the conduits of the present invention are hereincollectively termed “valves.”

One embodiment of the invention, therefore, provides a single-usecartridge with a sample-holding chamber connected to a first conduitwhich contains an analyte sensor or array of analyte sensors. A secondconduit, partly containing a fluid, is connected to the first conduitand air segments can be introduced into the fluid in the second conduitin order to segment it. Pump means are provided to displace the samplewithin the first conduit, and displaces fluid from the second conduitinto the first conduit. Thus, the sensor or sensors can be contactedfirst by a sample and then by a second fluid.

A second embodiment of the cartridge includes a closeable valve locatedbetween the first conduit and a waste chamber. This embodiment permitsdisplacement of the fluid from the second conduit into the first conduitusing only a single pump means connected to the first conduit. Thisembodiment further permits efficient washing of the conduits of thecartridge of the present invention, which is an important feature of asmall single-use cartridge. In operation, the sample is displaced tocontact the sensors, and is then displaced through the closeable valveinto the waste chamber. Upon wetting, the closeable valve seals theopening to the waste chamber, providing an airtight seal that allowsfluid in the second conduit to be drawn into contact with the sensorsusing only the pump means connected to the first conduit. In thisembodiment, the closeable valve permits the fluid to be displaced inthis manner and prevents air from entering the first conduit from thewaste chamber.

In another embodiment, both a closeable valve and means for introducingsegments into the conduit are provided. This embodiment has manyadvantages, among which is the ability to reciprocate a segmented fluidover the sensor or array of sensors. Thus a first segment or set ofsegments is used to rinse a sensor, and then a fresh segment replaces itfor taking measurements. Only one pump means (that connected to thefirst conduit) is required.

In a fourth embodiment analyte measurements are performed in a thin-filmof liquid coating an analyte sensor. Such thin-film determinations arepreferably performed amperometrically. This cartridge differs from theforegoing embodiments in having both a closeable valve that is sealedwhen the sample is expelled through the valve, and an air vent withinthe conduits that permits at least one air segment to be subsequentlyintroduced into the measuring fluid, thereby increasing the efficiencywith which the sample is rinsed from the sensor, and further permittingremoval of substantially all the liquid from the sensor prior tomeasurement, and still further permitting segments of fresh liquid to bebrought across the sensor to permit sequential, repetitive measurementsfor improved accuracy and internal checks of reproducibility.

The analysis scheme for the detection of low concentrations ofimmunoactive analyte relies on the formation of an enzyme labeledantibody/analyte/surface-bound antibody “sandwich” complex. Theconcentration of analyte in a sample is converted into a proportionalsurface concentration of an enzyme. The enzyme is capable of amplifyingthe analyte's chemical signal by converting a substrate to a detectableproduct. For example, where alkaline phosphatase is the enzyme, a singleenzyme molecule can produce about nine thousand detectable molecules perminute, providing several orders of magnitude improvement in thedetectability of the analyte compared to schemes in which anelectroactive species is attached to the antibody in place of alkalinephosphatase.

In immunosensor embodiments, it is advantageous to contact the sensorfirst with a sample and then with a wash fluid prior to recording aresponse from the sensor. In specific embodiments, the sample is amendedwith an antibody-enzyme conjugate that binds to the analyte of interestwithin the sample before the amended sample contacts the sensor. Bindingreactions in the sample produce an analyte/antibody-enzyme complex. Thesensor comprises an immobilized antibody to the analyte, attached closeto an electrode surface. Upon contacting the sensor, theanalyte/antibody-enzyme complex binds to the immobilized antibody nearthe electrode surface. It is advantageous at this point to remove fromthe vicinity of the electrode as much of the unbound antibody-enzymeconjugate as possible to minimize background signal from the sensor. Theenzyme of the antibody-enzyme complex is advantageously capable ofconverting a substrate, provided in the fluid, to produce anelectrochemically active species. This active species is produced closeto the electrode and provides either a current from a redox reaction atthe electrode when a suitable potential is applied (amperometricoperation). Alternatively, if the electroactive species is an ion, itcan be measured potentiometrically. In amperometric measurements thepotential may either be fixed during the measurement, or variedaccording to a predetermined waveform. For example, a triangular wavecan be used to sweep the potential between limits, as is used in thewell-known technique of cyclic voltammetry. Alternatively, digitaltechniques such as square waves can be used to improve sensitivity indetection of the electroactive species adjacent to the electrode. Fromthe current or voltage measurement, the amount or presence of theanalyte in the sample is calculated. These and other analyticalelectrochemical methods are well known in the art.

In embodiments in which the cartridge comprises an immunosensor, theimmunosensor is advantageously microfabricated from a base sensor of anunreactive metal such as gold, platinum or iridium, and a porouspermselective layer which is overlaid with a bioactive layer attached toa microparticle, for example latex particles. The microparticles aredispensed onto the porous layer covering the electrode surface, formingan adhered, porous bioactive layer. The bioactive layer has the propertyof binding specifically to the analyte of interest, or of manifesting adetectable change when the analyte is present, and is most preferably animmobilized antibody directed against the analyte.

In operation, therefore, one goal of the present invention is to providean immunosensor cartridge that is preferably operated in a basic senseas follows. (However, the invention is not restricted to embodimentscomprising an immunosensor, but includes any ligand-receptorinteraction, including complimentary strands of DNA and RNA,biotin-avidin and the like.) An unmetered amount of a preferablybiological sample is placed into the sample chamber of the cartridge,and the cartridge is placed into a reading apparatus. A metered portionof the sample is amended with at least one antibody-enzyme conjugate,and is then contacted with the immunosensor. A second fluid, whichcontains an electroinactive substrate for the enzyme, is used to rinsethe immunosensor substantially free of unbound antibody-enzymeconjugate, and the electrical response of the immunosensor electrode isrecorded and analyzed for the presence, or amount of, the analyte ofinterest. The cartridge may contain a plurality of immunosensors andreagents.

Signal Corrections

The calculation and correction methods can best be understood byreferring to FIGS. 23 and 24. A preferred embodiment is described withreference to a TnI cartridge cycle which involves amperometricmeasurements with two sensors, the cTnI sensor (amp0, ParamAct) whichbears the immunoassay reagent capable of specific binding of analyte tothe sensor surface, and a reference sensor (amp0, ParamRef) bearing animmunoassay reagent capable of specific binding of human serum albumin(HSA). The reference sensor becomes coated with HSA upon introduction ofthe sample and is used to assess and correct for non-specific binding ofsignal-generating reagent (conjugate). The net current is calculatedaccording to equation 5 (re-number equations below starting at 5) below;where the coefficient c0 is an optional manufacturing cartridgelot-specific value determined as the bias between amp0 and amp1 forun-spiked whole-blood and plasma, i.e., any bias in the absence ofanalyte.

iNet=ParamAct−ParamRef−c0(nanoamperes)  5

Various options exist for managing any temperature effect on animmunoassay of this type. For example, the assay can be run in a systemwhere the sample and other fluids and reagents are thermostated at agiven temperature, e.g., 37° C. Alternatively, the assay may be run atambient temperature, without any correction, or with correction to astandardized temperature based on measurement of the ambient value. Inanother embodiment, where a battery-powered analyzer is used and it isgenerally desirable to conserve battery life, it may be desirable toheat only the capture location of the assay device or cartridge. Herethe ambient temperature will have an effect on the cooling of the sampleif it enters regions adjacent to the capture location. In this example,the analyte capture and signal generation steps may have smalltemperature dependencies and so it is desirable that the net current iscorrected to an ambient temperature (ATemp), e.g., 23° C., for examplein accordance with equation 6. The value of the (per degree) coefficientc1 is generally not specific to a given lot of manufactured cartridges,but can be generalized to a given cartridge manufacturing process. Itgenerally has a relatively small value, e.g., 1 to 3%. One skilled inthe art will recognize that this can be determined fromtemperature-dependence experiments.

iTCorr=iNet*(1+c1*(ATemp−TempC))  6

It has been found that the amount of antigen captured and labeled on thesensor surface is dependent on the conductivity of the blood. Severalphenomena described below contribute to this aspect of the assay method.As described below, upon introduction to the cartridge the sample istreated with NaCl and other agents included in the sample holdingchamber, to reduce interferences related to white blood cells and othercomponents that can cause elevated readings in some samples. Thehypertonicity induced by the introduction of NaCl is observed to causeshrinkage of cells, red blood cells representing the greatest massfraction. As stated below, the amount of shrinkage is dependent on theplasma concentration of added NaCl, which is in turn dependent on theoriginal hematocrit (Hct) of the sample. Thus, the final hematocrit ofthe sample depends on the original hematocrit and the amount of saltadded. The conductivity of the sample after shrinkage depends on thefinal hematocrit and the final ionic strength.

The capacity for capture of signal generating reagents depends on theanalyte concentration and the conjugate concentration, both of whichbecome modified upon introduction of NaCl by virtue of the cellshrinkage. In the absence of added NaCl, the signal generation for aparticular TnI sample will increase (approximately linearly) withincreasing Hct. However, when addition of NaCl is factored into theequation, this linearity is transformed into quadratic behavior: as Hctincreases from 0, signal generation increases due to increasingconjugate concentration. However, as the Hct increases further, signalgeneration begins to decrease because the added NaCl causes increasingcell shrinkage and a corresponding decrease in conjugate and analyteconcentration. This is because the TnI (released from heart muscle) isrestricted to the plasma portion of the blood sample. As theconductivity is dependent on both the Hct and the ionic strength, it hasbeen found that the net signal generation is approximated by a quadraticfunction of the conductivity measured after cell shrinkage takes place.For this reason, it is desirable that the analytical signal is subjectedto a conductivity correction that corrects the net current to the valueexpected in plasma (Hct=0).

The conductivity correction function can be determined experimentally,where a whole blood sample is spiked with a known amount of TnI, andmanipulated through centrifugation and alteration of the plasma fractionso as to yield an array of standardized samples having the same plasmaconcentration of analyte but Hct varying from 0 to approximately 65percent. Performing an immunoassay of these samples in the cartridgesaffords collection of a set of signals as a function of conductivity.Normalization of this function, so that plasma is associated with asignal generation factor of unity, affords the conductivity correctionfunction. This may take one of several mathematical forms, includingfour point logistical functions and the quadratic form shown in equation7. In this example, ResHct is the resistance at the hematocrit(conductivity) sensor after the sample has been amended by the reagent.

fCond=c2*ResHct² +c3*ResHct+c4  7

One skilled in the art will recognize that the actual coefficients inEquation 7 will depend on the reagent components and have somesensitivity to the means by which the immunoassay is carried out, e.g.,the capture time. In one embodiment, the value of fCond is limited byequations 8 and 9. In equation 8, if the measured conductivity is belowa value expected for a Hct value of for example 15 percent, then thesample is treated as a plasma sample and the correction factor is set to5. Equation 9 says that if the sample is clearly not a plasma sample andthe correction factor is smaller than 0.8, then limit the correction to0.8. Note that it is straightforward to find that the maximum value offCond occurs when ResHct=−½*(c3/c2).

If ResHct<MaxPlasmaCond set fCond=1  8

If ResHct>MaxPlasmaCond and fCond<MinfCond, set fCond=MinfCond  9

Where MaxPlasmaCond=1050 and MinfCond=0.8

The correction factor as determined via equations 7-9 is applied asdefined in equation 10.

iCorr=iTCorr/fCond  10

The analyte concentration is then calculated, for example by one ofequations 11-13 below. In one embodiment, the cartridge is provided witha barcode with factory set information including the equations to beused and the required test coefficients. The analyzer, into which thecartridge is inserted to run the test, is thus equipped with a barcodereader. A selection of equations may be embedded in the software of theanalyzer. For example, the coefficients for the cartridge may differ,where different lots of cartridges are manufactured, each lot havingslightly different factory-determined characteristics. In any event, thecoefficients for the cartridge, from whichever manufacturing lot thecartridge is drawn, are conveyed to the analyzer for use in one or moreof the equations, for that particular cartridge test. For example, if agiven digit of the cartridge barcode is set to 1, the analyzer may setc6 to zero, whereas other digits may code for different coefficients orselect a kinetic model to be used, e.g., an immunoassay model formulatedby analogy to the well-known Michaelis-Menton enzyme kinetics, as inequation 11.

$\begin{matrix}{{\lbrack{cTnl}\rbrack \left( {{ng}/{mL}} \right)} = {c\; 6^{*}\; {{Corr}/\left( {{c\; 5^{*}c\; 6} - {\; {Corr}}} \right)}}} & 11 \\{{\lbrack{cTnl}\rbrack \left( {{ng}/{mL}} \right)} = {{c\; 7^{*}\; {Corr}^{2}} + {c\; 7^{*}c\; 8^{*}\; {Corr}}}} & 12 \\\begin{matrix}{{\lbrack{cTnl}\rbrack \left( {{ng}/{mL}} \right)} = {{c\; 6^{*}\; {{Corr}/\left( {{c\; 5^{*}c\; 6} - {\; {Corr}}} \right)}} +}} \\{{{c\; 7^{*}\; {Corr}^{2}} + {c\; 7^{*}c\; 8^{*}\; {Corr}}}} \\{= {{c\; 6^{*}\; {{Corr}/\left( {{c\; 5^{*}c\; 6} - {\; {Corr}}} \right)}} +}} \\{{{c\; 7^{*}\; {Corr}^{2}} + {{Linear}^{*}\; {Corr}}}}\end{matrix} & 13\end{matrix}$

In the event that the Michaelis-Menton model is employed, an additionallimit may be imposed, for example as defined by equation 12.

If iCorr>0.9*c5*c6, set iCorr=0.9*c5*c6  14

In addition, as a practical matter in reporting an analyte value to theuser, (typically a physician by means of a display screen on theanalyzer), in a given range from zero to an upper maximum value, oneskilled in the art will recognize that the maximum theoretical currentthat can be observed is equal to the product c5*c6. Thus it is desirablethat iCorr is limited to 90% of this value (equation 14) in order toavoid approaching the point of discontinuity inherent in theMichaelis-Menton term, i.e., when iCorr=c5*c6. As a result, the reportedvalue can be limited to a given range. In the troponin cartridgeexample, the reported [cTnI] value is thus restricted to the range equalto or greater than zero and less than or equal to 50 ng/mL.

Cartridge Construction

Referring to the Figures, the cartridge of the present inventioncomprises a cover, FIGS. 1, 2, a base, FIG. 4, and a thin-film adhesivegasket, FIG. 3, disposed between the base and the cover. Referring nowto FIG. 1, the cover 1 is made of a rigid material, preferably plastic,and capable of repetitive deformation at flexible hinge regions 5, 9, 10without cracking. The cover comprises a lid 2, attached to the main bodyof the cover by a flexible hinge 9. In operation, after introduction ofa sample into the sample holding chamber 34, the lid can be secured overthe entrance to the sample entry port 4, preventing sample leakage, andthe lid is held in place by hook 3. The cover further comprises twopaddles 6, 7, that are moveable relative to the body of the cover, andwhich are attached to it by flexible hinge regions 5, 10. In operation,when operated upon by a pump means, paddle 6 exerts a force upon an airbladder comprised of cavity 43, which is covered by thin-film gasket 21,to displace fluids within conduits of the cartridge. When operated by asecond pump means, paddle 7 exerts a force upon the gasket 21, which candeform because of slits 22 cut therein. The cartridge is adapted forinsertion into a reading apparatus, and therefore has a plurality ofmechanical and electrical connections for this purpose. It should alsobe apparent that manual operation of the cartridge is possible. Thus,upon insertion of the cartridge into a reading apparatus, the gaskettransmits pressure onto a fluid-containing foil pack filled withapproximately 130 uL of analysis/wash solution (“fluid”) located incavity 42, rupturing the package upon spike 38, and expelling fluid intoconduit 39, which is connected via a short transecting conduit in thebase to the sensor conduit. The analysis fluid fills the front of theanalysis conduit first pushing fluid onto a small opening in the tapegasket that acts as a capillary stop. Other motions of the analyzermechanism applied to the cartridge are used to inject one or moresegments into the analysis fluid at controlled positions within theanalysis conduit. These segments are used to help wash the sensorsurface and the surrounding conduit with a minimum of fluid.

The cover further comprises a hole covered by a thin pliable film 8. Inoperation, pressure exerted upon the film expels one or more airsegments into a conduit 20 through a small hole 28 in the gasket.

Referring to FIG. 2, the lower surface of the base further comprisessecond conduit 11, and first conduit 15. Second conduit 11 includes aconstriction 12, which controls fluid flow by providing resistance tothe flow of a fluid. Optional coatings 13, 14 provide hydrophobicsurfaces, which together with gasket holes 31, 32, control fluid flowbetween conduits 11, 15. A recess 17 in the base provides a pathway forair in conduit 34 to pass to conduit 34 through hole 27 in the gasket.

Referring to FIG. 3, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Thus, hole 24 permits fluid to flow from conduit 11 intowaste chamber 44; hole 25 comprises a capillary stop between conduits 34and 11; hole 26 permits air to flow between recess 18 and conduit 40;hole 27 provides for air movement between recess 17 and conduit 34; andhole 28 permits fluid to flow from conduit 19 to waste chamber 44 viaoptional closeable valve 41. Holes 30 and 33 permit the plurality ofelectrodes that are housed within cutaways 35 and 37, respectively, tocontact fluid within conduit 15. In a specific embodiment, cutaway 37houses a ground electrode, and/or a counter-reference electrode, andcutaway 35 houses at least one analyte sensor and, optionally, aconductimetric sensor.

Referring to FIG. 4, conduit 34 is the sample holding chamber thatconnects the sample entry port 4 to first conduit 11 in the assembledcartridge. Cutaway 35 houses the analyte sensor or sensors, or ananalyte responsive surface, together with an optional conductimetricsensor or sensors. Cutaway 37 houses a ground electrode if needed as areturn current path for an electrochemical sensor, and may also house anoptional conductimetric sensor. Cutaway 36 provides a fluid path betweengasket holes 31 and 32 so that fluid can pass between the first andsecond conduits. Recess 42 houses a fluid-containing package, e.g., arupturable pouch, in the assembled cartridge that is pierced by spike 38because of pressure exerted upon paddle 7 upon insertion into a readingapparatus. Fluid from the pierced package flows into the second conduitat 39. An air bladder is comprised of recess 43 which is sealed on itsupper surface by gasket 21. The air bladder is one embodiment of a pumpmeans, and is actuated by pressure applied to paddle 6 which displacesair in conduit 40 and thereby displaces the sample from sample chamber34 into first conduit 15.

The location at which air enters the sample chamber (gasket hole 27)from the bladder, and the capillary stop 25, together define apredetermined volume of the sample chamber. An amount of the samplecorresponding to this volume is displaced into the first conduit whenpaddle 6 is depressed. This arrangement is therefore one possibleembodiment of a metering means for delivering a metered amount of anunmetered sample into the conduits of the cartridge.

In the present cartridge, a means for metering a sample segment isprovide in the base plastic part. The segment size is controlled by thesize of the compartment in the base and the position of the capillarystop and air pipe holes in the tape gasket. This volume can be readilyvaried from 2 to 200 microliters. Expansion of this range of samplesizes is possible within the context of the present invention.

The fluid is pushed through a pre-analytical conduit 11 that can be usedto amend a reagent (e.g., particles or soluble molecules) into thesample prior to its presentation at the sensor conduit 19.Alternatively, the amending reagent may be located in portion 15, beyondportion 16. Pushing the sample through the pre-analytical conduit alsoserves to introduce tension into the diaphragm pump paddle 7 whichimproves its responsiveness for actuation of fluid displacement.

In some assays, metering is advantageous if quantitation of the analyteis required. A waste chamber is provided, 44, for sample and/or fluidthat is expelled from the conduit, to prevent contamination of theoutside surfaces of the cartridge. A vent connecting the waste chamberto the external atmosphere is also provided, 45. A feature of thecartridge is that once a sample is loaded, analysis can be completed andthe cartridge discarded without the operator or others contacting thesample.

Referring now to FIG. 5, a schematic diagram of the features of acartridge and components is provided, wherein 51-57 are portions of theconduits and sample chamber that can optionally be coated with dryreagents to amend a sample or fluid. The sample or fluid is passed atleast once over the dry reagent to dissolve it. Reagents used to amendsamples or fluid within the cartridge include antibody-enzymeconjugates, or blocking agents that prevent either specific ornon-specific binding reactions among assay compounds. A surface coatingthat is not soluble but helps prevent non-specific adsorption of assaycomponents to the inner surfaces of the cartridges can also be provided.

Within a segment of sample or fluid, an amending substance can bepreferentially dissolved and concentrated within a predetermined regionof the segment. This is achieved through control of the position andmovement of the segment. Thus, for example, if only a portion of asegment, such as the leading edge, is reciprocated over the amendedsubstance, then a high local concentration of the substance can beachieved close to the leading edge. Alternatively, if an homogenousdistribution of the substance is desired, for example if a knownconcentration of an amending substance is required for a quantitativeanalysis, then further reciprocation of the sample or fluid will resultin mixing and an even distribution.

In specific embodiments, a closeable valve is provided between the firstconduit and the waste chamber. In one embodiment, this valve, 58, iscomprised of a dried sponge material that is coated with an impermeablesubstance. In operation, contacting the sponge material with the sampleor a fluid results in swelling of the sponge to fill the cavity 41,thereby substantially blocking further flow of liquid into the wastechamber 44. Furthermore, the wetted valve also blocks the flow of airbetween the first conduit and the waste chamber, which permits the firstpump means connected to the sample chamber to displace fluid within thesecond conduit, and to displace fluid from the second conduit into thefirst conduit in the following manner. After the sample is exposed tothe sensor for a controlled time, the sample is moved into thepost-analytical conduit 19 where it can be amended with another reagent.It can then be moved back to the sensor and a second reaction period canbegin. Alternately, the post-analysis conduit can serve simply toseparate the sample segment from the sensor. Within this post-analysisconduit is a single closeable valve which connects the air vent of thesensor conduit to the diaphragm air pump. When this valve closes, thesample is locked in the post analytical conduit and cannot be moved backto the sensor chip. There are several different design examples for thisvalve that are encompassed within the present invention. Some designsare activated mechanically while others activate on liquid contact.Other types of closeable valve that are encompassed by the presentinvention include, but are not limited to; a flexible flap held in anopen position by a soluble glue or a gelling polymer that dissolves orswells upon contact with a fluid or sample thus causing the flap toclose; and alternatively, in one specific embodiment, a thin layer of aporous paper or similar material interposed between a conduit and eitherthe waste chamber or ambient air such that the paper is permeable to airwhile dry but impermeable when wet. In the latter case it is notnecessary that the closeable valve be interposed between a conduit andthe waste chamber: the valve passes little to no liquid before closingand so the valve is appropriately placed when positioned between aconduit and the ambient air surrounding the cartridge. In practicalconstruction, a piece of filter paper is placed on an opening in thetape gasket in the fluid path to be controlled. Air can readily movethrough this media to allow fluid to be moved through the fluid path.When the fluid is pushed over this filter, the filter media becomesfilled with liquid and further motion through the fluid path is stopped.Once the filter become wet, significant pressures would be required tomove liquid through the pores of the filter. Air flow through the filteris also prevented because of the higher pressure required to push theliquid out of the filter, typically termed bubble pressure. This valveembodiment requires very little liquid to actuate the valve, andactuation occurs rapidly and reliably. Materials, their dimensions,porosity, wettability, swelling characteristics and related parametersare selected to provide for rapid closure, within one second or moreslowly, e.g., up to 60 seconds, after first contacting the sample,depending on the specific desired closure time.

Alternatively, the closeable valve is a mechanical valve. In thisembodiment, a latex diaphragm is placed in the bottom of the air bladderon top of a specially constructed well. The well contains two openingswhich fluidically connect the air vent to the sample conduit. As theanalyzer plunger pushes to the bottom of the air bladder, it presses onthis latex diaphragm which is adhesive backed and seals the connectionbetween the two holes. This blocks the sample's air vent, locking thesample in place.

Referring now to FIG. 6, which illustrates the schematic layout of animmunosensor cartridge, there are provided three pump means, 61-63.While these pumps have been described in terms of specific embodiments,it will be readily understood that any pump means capable of performingthe respective functions of pump means 61-63 may be used within thepresent invention. Thus, pump means 1, 61, must be capable of displacingthe sample from the sample holding chamber into the first conduit; pumpmeans 2, 62, must be capable of displacing fluid within the secondconduit; and pump means 3, 63, must be capable of inserting at least onesegment into the second conduit. Other types of pump which are envisagedin the present application include, but are not limited to, an air saccontacting a pneumatic means whereby pressure is applied to said airsac, a flexible diaphragm, a piston and cylinder, an electrodynamicpump, and a sonic pump. With reference to pump means 3, 63, the term“pump means” includes all methods by which one or more segments areinserted into the second conduit, such as a pneumatic means fordisplacing air from an air sac, a dry chemical that produces a gas whendissolved, or a plurality of electrolysis electrodes operably connectedto a current source. In a specific embodiment, the segment is producedusing a mechanical segment generating diaphragm that may have more thanone air bladder or chamber. The well 8 has a single opening whichconnects the inner diaphragm pump and the fluid filled conduit intowhich a segment is to be injected 20. The diaphragm can be segmented toproduce multiple segments, each injected in a specific location within afluid filled conduit.

In alternative embodiments, a segment is injected using a passivefeature. A well in the base of the cartridge is sealed by tape gasket.The tape gasket covering the well has two small holes on either end. Onehole is open while the other is covered with a filter material whichwets upon contact with a fluid. The well is filled with a loosehydrophilic material such as a cellulose fiber filter, paper filter orglass fiber filter. This hydrophilic material draws the liquid into thewell in the base via capillary action, displacing the air which wasformerly in the well. The air is expelled through the opening in thetape gasket creating a segment whose volume is determined by the volumeof the well and the void volume of the loose hydrophilic material. Thefilter used to cover one of the inlets to the well in the base can bechosen to meter the rate at which the fluid fills the well and therebycontrol the rate at which the segment is injected into the conduit inthe cover. This passive feature permits any number of controlledsegments to be injected at specific locations within a fluid path andrequires a minimum of space.

The present invention will be better understood with reference to thespecific embodiments set forth in the following examples.

EXAMPLE 1

Referring now to FIG. 7, which illustrates the principle of anamperometric immunoassay according to specific embodiments of thepresent invention for determination of troponin I (TnI), a marker ofcardiac function. A blood sample, for example, is introduced into thesample holding chamber of a cartridge of the present invention, and isamended by a conjugate molecule comprising alkaline phosphatase enzyme(AP) covalently attached to a polyclonal anti-troponin I antibody (aTnI)71. This conjugate specifically binds to the TnI, 70, in the bloodsample, producing a complex made up of TnI bound to the AP-aTnIconjugate. In a capture step, this complex binds to the capture aTnIantibody 72 attached on, or close to, the immunosensor. The sensor chiphas a conductivity sensor which is used to monitor when the samplereaches the sensor chip. The time of arrival of the fluid can be used todetect leaks within the cartridge: a delay in arrival signals a leak.The position of the sample segment within the sensor conduit can beactively controlled using the edge of the fluid as a marker. As thesample/air interface crosses the conductivity sensor, a precise signalis generated which can be used as a fluid marker from which controlledfluid excursions can be executed. The fluid segment is preferentiallyoscillated edge-to-edge over the sensor in order to present the entiresample to the sensor surface. A second reagent can be introduced in thesensor conduit beyond the sensor chip, which becomes homogenouslydistributed during the fluid oscillations.

The sensor chip contains a capture region or regions coated withantibodies for the analyte of interest. These capture regions aredefined by a hydrophobic ring of polyimide or anotherphotolithographically produced layer. A microdroplet or severalmicrodroplets (approximately 5-40 nanoliters in size) containingantibodies in some form, for example bound to latex microspheres, isdispensed on the surface of the sensor. The photodefined ring containsthis aqueous droplet allowing the antibody coated region to be localizedto a precision of a few microns. The capture region can be made from0.03 to roughly 2 square millimeters in size. The upper end of this sizeis limited by the size of the conduit and sensor in present embodiments,and is not a limitation of the invention.

Thus, the gold electrode 74 is coated with a biolayer 73 comprising acovalently attached anti-troponin I antibody, to which the TnI/AP-aTnIcomplex binds. AP is thereby immobilized close to the electrode inproportion to the amount of TnI initially present in the sample. Inaddition to specific binding, the enzyme-antibody conjugate may bindnon-specifically to the sensor. Non-specific binding provides abackground signal from the sensor that is undesirable and preferably isminimized. As described above, the rinsing protocols, and in particularthe use of segmented fluid to rinse the sensor, provide efficient meansto minimize this background signal. In a second step subsequent to therinsing step, a substrate 75 that is hydrolyzed by, for example,alkaline phosphatase to produce an electroactive product 76 is presentedto the sensor. In specific embodiments the substrate is comprised of aphosphorylated ferrocene or p-aminophenol. The amperometric electrode iseither clamped at a fixed electrochemical potential sufficient tooxidize or reduce a product of the hydrolyzed substrate but not thesubstrate directly, or the potential is swept one or more times throughan appropriate range. Optionally, a second electrode may be coated witha layer where the complex of TnI/AP-aTnI is made during manufacture, toact as a reference sensor or calibration means for the measurement.

In the present example, the sensor comprises two amperometric electrodeswhich are used to detect the enzymatically produced 4-aminophenol fromthe reaction of 4-aminophenylphosphate with the enzyme label alkalinephosphatase. The electrodes are preferably produced from gold surfacescoated with a photodefined layer of polyimide. Regularly spaced openingin the insulating polyimide layer define a grid of small gold electrodesat which the 4-aminophenol is oxidized in a 2 electron per moleculereaction. Sensor electrodes further comprise a biolayer, while referenceelectrodes can be constructed, for example, from gold electrodes lackinga biolayer, or from silver electrodes, or other suitable material.Different biolayers can provide each electrode with the ability to sensea different analyte.

H₂N—C₆H₄—OH—->HN═C₆H₄═O+2H⁺+2e ⁻

Substrates, such as p-aminophenol species, can be chosen such that theE_(1/2) of the substrate and product differ substantially. Preferably,the voltammetric half-wave potential (E_(1/2)) of the substrate issubstantially higher (more positive) than that of the product. When thecondition is met, the product can be selectively electrochemicallymeasured in the presence of the substrate.

The size and spacing of the electrode play an important role indetermining the sensitivity and background signal. The importantparameters in the grid are the percentage of exposed metal and thespacing between the active electrodes. The position of the electrode canbe directly underneath the antibody capture region or offset from thecapture region by a controlled distance. The actual amperometric signalof the electrodes depends on the positioning of the sensors relative tothe antibody capture site and the motion of the fluid during theanalysis. A current at the electrode is recorded that depends upon theamount of electroactive product in the vicinity of the sensor.

The detection of alkaline phosphatase activity in this example relies ona measurement of the 4-aminophenol oxidation current. This is achievedat a potential of about +60 mV versus the Ag/AgCl ground chip. The exactform of detection used depends on the sensor configuration. In oneversion of the sensor, the array of gold microelectrodes is locateddirectly beneath the antibody capture region. When the analysis fluid ispulled over this sensor, enzyme located on the capture site converts the4-aminophenylphosphate to 4-aminophenol in an enzyme limited reaction.The concentration of the 4-aminophenylphosphate is selected to be inexcess, e.g., 10 times the Km value. The analysis solution is 0.1 M indiethanolamine, 1.0 M NaCl, buffered to a pH of 9.8. Additionally, theanalysis solution contains 0.5 mM MgCl which is a cofactor for theenzyme. Alternatively, a carbonate buffer has the desired properties.

In another electrode geometry embodiment, the electrode is located a fewhundred microns away from the capture region. When a fresh segment ofanalysis fluid is pulled over the capture region, the enzyme productbuilds with no loss due to electrode reactions. After a time, thesolution is slowly pulled from the capture region over the detectorelectrode resulting in a current spike from which the enzyme activitycan be determined.

An important consideration in the sensitive detection of alkalinephosphatase activity is the non-4-aminophenol current associated withbackground oxidations and reductions occurring at the gold sensor. Goldsensors tend to give significant oxidation currents in basic buffers atthese potentials. The background current is largely dependent on thebuffer concentration, the area of the gold electrode (exposed area),surface pretreatments and the nature of the buffer used. Diethanolamineis a particularly good activating buffer for alkaline phosphatase. Atmolar concentrations, the enzymatic rate is increased by about threetimes over a non-activating buffer such as carbonate.

In alternative embodiments, the enzyme conjugated to an antibody orother analyte-binding molecule is urease, and the substrate is urea.Ammonium ions produced by the hydrolysis of urea are detected in thisembodiment by the use of an ammonium sensitive electrode.Ammonium-specific electrodes are well-known to those of skill in theart. A suitable microfabricated ammonium ion-selective electrode isdisclosed in U.S. Pat. No. 5,200,051, incorporated herein by reference.Other enzymes that react with a substrate to produce an ion are known inthe art, as are other ion sensors for use therewith. For example,phosphate produced from an alkaline phosphatase substrate can bedetected at a phosphate ion-selective electrode.

Referring now to FIG. 8, there is illustrated the construction of anembodiment of a microfabricated immunosensor. Preferably a planarnon-conducting substrate is provided, 80, onto which is deposited aconducting layer 81 by conventional means or microfabrication known tothose of skill in the art. The conducting material is preferably a noblemetal such as gold or platinum, although other unreactive metals such asiridium may also be used, as may non-metallic electrodes of graphite,conductive polymer, or other materials. An electrical connection 82 isalso provided. A biolayer 83 is deposited onto at least a portion of theelectrode. In the present disclosure, a biolayer means a porous layercomprising on its surface a sufficient amount of a molecule 84 that caneither bind to an analyte of interest, or respond to the presence ofsuch analyte by producing a change that is capable of measurement.Optionally, a permselective screening layer may be interposed betweenthe electrode and the biolayer to screen electrochemical interferents asdescribed in U.S. Pat. No. 5,200,051.

In specific embodiments, a biolayer is constructed from latex beads ofspecific diameter in the range of about 0.001 to 50 microns. The beadsare modified by covalent attachment of any suitable molecule consistentwith the above definition of a biolayer. Many methods of attachmentexist in the art, including providing amine reactiveN-hydroxysuccinimide ester groups for the facile coupling of lysine orN-terminal amine groups of proteins. In specific embodiments, thebiomolecule is chosen from among ionophores, cofactors, polypeptides,proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,lectins, neurochemical receptors, oligonucleotides, polynucleotides,DNA, RNA, or suitable mixtures. In most specific embodiments, thebiomolecule is an antibody selected to bind one or more of humanchorionic gonadotrophin, troponin I, troponin T, troponin C, a troponincomplex, creatine kinase, creatine kinase subunit M, creatine kinasesubunit B, myoglobin, myosin light chain, or modified fragments ofthese. Such modified fragments are generated by oxidation, reduction,deletion, addition or modification of at least one amino acid, includingchemical modification with a natural moiety or with a synthetic moiety.Preferably, the biomolecule binds to the analyte specifically and has anaffinity constant for binding analyte ligand of about 10⁷ to 10¹⁵ M⁻¹.

In one embodiment, the biolayer, comprising beads having surfaces thatare covalently modified by a suitable molecule, is affixed to the sensorby the following method. A microdispensing needle is used to depositonto the sensor surface a small droplet, preferably about 20 nL, of asuspension of modified beads. The droplet is permitted to dry, whichresults in a coating of the beads on the surface that resistsdisplacement during use.

In addition to immunosensors in which the biolayer is in a fixedposition relative to an amperometric sensor, the present invention alsoenvisages embodiments in which the biolayer is coated upon particlesthat are mobile. The cartridge can contain mobile microparticles capableof interacting with an analyte, for example magnetic particles that arelocalized to an amperometric electrode subsequent to a capture step,whereby magnetic forces are used to concentrate the particles at theelectrode for measurement. One advantage of mobile microparticles in thepresent invention is that their motion in the sample or fluidaccelerates binding reactions, making the capture step of the assayfaster. For embodiments using non-magnetic mobile microparticles, aporous filter is used to trap the beads at the electrode.

Referring now to FIG. 9, there is illustrated a mask design for severalelectrodes upon a single substrate. By masking and etching techniques,independent electrodes and leads can be deposited. Thus, a plurality ofimmunosensors, 94 and 96, and conductimetric sensors, 90 and 92, areprovided in a compact area at low cost, together with their respectiveconnecting pads, 91, 93, 95, and 97, for effecting electrical connectionto the reading apparatus. In principle, a very large array of sensorscan be assembled in this way, each sensitive to a different analyte oracting as a control sensor or reference immunosensor.

Specifically, immunosensors are prepared as follows. Silicon wafers arethermally oxidized to form approximately a 1 micron insulating oxidelayer. A titanium/tungsten layer is sputtered onto the oxide layer to apreferable thickness of between 100-1000 Angstroms, followed by a layerof gold that is most preferably 800 Angstroms thick. Next, a photoresistis spun onto the wafer and is dried and baked appropriately. The surfaceis then exposed using a contact mask, such as a mask corresponding tothat illustrated in FIG. 9. The latent image is developed, and the waferis exposed to a gold-etchant. The patterned gold layer is coated with aphotodefinable polyimide, suitably baked, exposed using a contact mask,developed, cleaned in an O₂ plasma, and preferably imidized at 350° C.for 5 hours. An optional metallization of the back side of the wafer maybe performed to act as a resistive heating element, where theimmunosensor is to be used in a thermostatted format. The surface isthen printed with antibody-coated particles. Droplets, preferably ofabout 20 nL volume and containing 1% solid content in deionized water,are deposited onto the sensor region and are dried in place by airdrying. Optionally, an antibody stabilization reagent (supplied bySurModica Corp. or AET Ltd.) is overcoated onto the sensor.

Drying the particles causes them to adhere to the surface in a mannerthat prevents dissolution in either sample or fluid containing asubstrate. This method provides a reliable and reproducibleimmobilization process suitable for manufacturing sensor chips in highvolume.

Referring now to FIG. 10, there are illustrated results obtained foranalysis of samples containing 0 or 50 miU/mL human chorionicgonadotrophin (HCG) and an HCG-sensitive amperometric immunosensor. Attime 100, a solution containing a p-aminophenol phosphate is supplied toa sensor which is previously treated with HCG and an anti-HCG polyclonalantibody conjugated to alkaline phosphatase. As the substrate ishydrolyzed by alkaline phosphatase, a current increases to a maximum101, and thereafter declines 102, as substrate within the diffusionvolume of the sensor is depleted and oxidized p-aminophenol accumulates.Good reproducibility is obtained between sensors, as shown by the outputsignal characteristics of individual single-use sensors. In operation,displacement of the fluid containing the enzyme substrate provides freshsubstrate to the electrode surface, and also removes products, so thatmultiple readings are easily obtained for a single sample. In analternative embodiment, the signal at the electrode is augmented byenzymatic regeneration of the electroactive species in the vicinity ofthe electrode. In a specific embodiment, a phosphorylated ferrocene isused as the substrate for alkaline phosphatase attached to the antibody.Hydrolysis yields a ferrocene product, which is oxidized and detected atthe electrode. In a second step, glucose oxidase enzyme and glucose areused to re-reduce the electrochemically oxidized ferrocene, with aconsequent increase in the current and detection sensitivity. Referringnow to FIG. 13, an electrode 130 oxidizes or reduces the electroactiveproduct 132 of alkaline phosphatase immobilized as a complex 131 on orclose to the electrode surface. In a second step, the electroactivespecies 132 is regenerated from the product 133 by the catalytic actionof enzyme 134. This cycling reaction increases the concentration ofelectroactive species 132 in proximity to the electrode surface 130, andthereby increases the current recorded at the electrode.

Referring now to FIG. 11, there is shown dose-response results obtainedusing HCG and an HCG-responsive amperometric immunosensor. Amounts ofHCG equivalent to 0 to 50 miU/mL are allowed to bind to the immobilizedantibody attached to the electrode, as in FIG. 10. Referring now to FIG.12, good linearity, and 121, of the response of the peak sensor currentwith increasing HCG is found. Thus, it is demonstrated that thisembodiment can precisely and rapidly quantify HCG in a sample.

EXAMPLE 2 Method of Use of a Cartridge

An unmetered fluid sample is introduced into sample chamber 34 of acartridge according to claim 1, through sample entry port 4. Capillarystop 25 prevents passage of the sample into conduit 11 at this stage,and conduit 34 is filled with the sample. Lid 2 or element 200 is closedto prevent leakage of the sample from the cartridge. The cartridge isthen inserted into a reading apparatus, such as that disclosed in U.S.Pat. No. 5,821,399 to Zelin, which is hereby incorporated by reference.Insertion of the cartridge into a reading apparatus activates themechanism which punctures a fluid-containing package located at 42 whenthe package is pressed against spike 38. Fluid is thereby expelled intothe second conduit, arriving in sequence at 39, 20, 12 and 11. Theconstriction at 12 prevents further movement of fluid because residualhydrostatic pressure is dissipated by the flow of fluid via secondconduit portion 11 into the waste chamber 44. In a second step,operation of a pump means applies pressure to air-bladder 43, forcingair through conduit 40, through cutaways 17 and 18, and into conduit 34at a predetermined location 27. Capillary stop 25 and location 27delimit a metered portion of the original sample. While the sample iswithin sample chamber 34, it is optionally amended with a compound orcompounds present initially as a dry coating on the inner surface of thechamber. The metered portion of the sample is then expelled through thecapillary stop by air pressure produced within air bladder 43. Thesample passes into conduit 15 and into contact with the analyte sensoror sensors located within cutaway 35.

In embodiments employing an immunosensor located within cutout 35, thesample is amended prior to arriving at the sensor by, for example, anenzyme-antibody conjugate. An antibody that binds the analyte ofinterest is covalently attached to an enzyme that can generate a redoxactive substance close to an amperometric electrode. In specificembodiments, the enzyme may be alkaline phosphatase, which hydrolyzescertain organophosphate compounds, such as derivatives of p-aminophenolthat liberate redox-active compounds when hydrolyzed. However, anyenzyme capable of producing, destroying, or altering any compound thatmay be detected by a sensor may be employed in conjunction with amatching sensor. For example, antibody-urease conjugate may be usedtogether with an ammonium sensor. Thus, the enzyme-antibody conjugate orconjugates amends the sample and binds to the analyte of interest. Theimmunosensor can comprise immobilized antibody that binds to an analyteof interest. When the amended sample passes over the immunosensor, theanalyte of interest binds to the sensor, together with antibody-enzymeconjugate to which it is attached.

To promote efficient binding of the analyte to the sensor, the samplecontaining the analyte is optionally passed repeatedly over the sensorin an oscillatory motion. Preferably, an oscillation frequency ofbetween about 0.2 and 2 Hz is used, most preferably 0.7 Hz. Thus enzymeis brought into close proximity to the amperometric electrode surface inproportion to the amount of analyte present in the sample.

Once an opportunity for the analyte/enzyme-antibody conjugate complex tobind to the immunosensor has been provided, the sample is ejected byfurther pressure applied to air bladder 43, and the sample passes towaste chamber 44.

A wash step next removes non-specifically bound enzyme-conjugate fromthe sensor chamber. Fluid in the second conduct is moved by a pump means43, into contact with the sensors. The analysis fluid is pulled slowlyuntil the first air segment is detected at a conductivity sensor.

The air segment or segment can be produced within a conduit by anysuitable means, including but not limited to, passive means, as shown inFIG. 14 and described below; active means including a transient loweringof the pressure within a conduit using pump means whereby air is drawninto the conduit through a flap or valve; or by dissolving a compoundpre-positioned within a conduit that liberates a gas upon contactingfluid in the conduit, where such compound may include a carbonate,bicarbonate or the like. This segment is extremely effective at clearingthe sample-contaminated fluid from conduit 15. The efficiency of therinsing of the sensor region is greatly enhanced by the introduction ofone or more air segments into the second conduit as described. Theleading and/or trailing edges of air segments are passed one or moretimes over the sensors to rinse and resuspend extraneous material thatmay have been deposited from the sample. Extraneous material includesany material other than specifically bound analyte oranalyte/antibody-enzyme conjugate complex. However, it is an object ofthe invention that the rinsing is not sufficiently protracted orvigorous as to promote dissociation of specifically bound analyte oranalyte/antibody-enzyme conjugate complex from the sensor.

A second advantage of introducing air segments into the fluid is tosegment the fluid. For example, after a first segment of the fluid isused to rinse a sensor, a second segment is then placed over the sensorwith minimal mixing of the two segments. This feature further reducesbackground signal from the sensor by more efficiently removing unboundantibody-enzyme conjugate. After the front edge washing, the analysisfluid is pulled slowly until the first air segment is detected at aconductivity sensor. This segment is extremely effective at clearing thesample-contaminated fluid which was mixed in with the first analysisfluid sample.

A second advantage of introducing air segments into conduit two is tosegment the fluid. For example, after a first segment of the fluid isused to rinse a sensor, a second segment is then placed over the sensorwith minimal mixing of the two segments. This feature further reducesbackground signal from the sensor by more efficiently removing unboundantibody-enzyme conjugate.

For measurement, a new portion of fluid is placed over the sensors, andthe current or potential, as appropriate to the mode of operation, isrecorded as a function of time.

EXAMPLE 3 Method of Use of a Cartridge

Use of a cartridge with a closeable valve, preferably located betweenthe sensor chamber and the waste chamber, is herein illustrated by aspecific embodiment in which the concentration of HCG is determinedwithin a blood sample, which is introduced into the sample chamber ofsaid cartridge. In the following time sequence, time zero (t=0)represents the time at which the cartridge is inserted into thecartridge reading device. Times are given in minutes. Between t=0 andt=1.5, the cartridge reading device makes electrical contact with thesensors through pads 91, 93, 95, and 97, and performs certain diagnostictests. Insertion of the cartridge perforates the foil pouch introducingfluid into the second conduit as previously described. The diagnostictests determine whether fluid or sample is present in the conduits usingthe conductivity electrodes; determine whether electrical short circuitsare present in the electrodes; and ensure that the sensor and groundelectrodes are thermally equilibrated to, preferably, 37° C. prior tothe analyte determination.

Between t=1.5 and t=6.75, a metered portion of the sample, preferablybetween 4 and 200 μl, more preferably between 4 and 20 μl, and mostpreferably 7 μl, is used to contact the sensor as described in EXAMPLE2. The edges defining the forward and trailing edges of the sample arereciprocally moved over the sensor region at a frequency that ispreferably between 0.2 to 2.0 Hz, and is most preferably 0.7 Hz. Duringthis time, the enzyme-antibody conjugate dissolves within the sample, aspreviously described. The amount of enzyme-antibody conjugate that iscoated onto the conduit is selected to yield a concentration whendissolved that is preferably higher than the highest anticipated HCGconcentration, and is most preferably six times higher than the highestanticipated HCG concentration in the sample.

Between t=6.75 and t=10.0 the sample is moved into the waste chamber viacloseable valve 41, wetting the closeable valve and causing it to closeas previously described. The seal created by the closing of the valvepermits the first pump means to be used to control motion of fluid fromconduit 11 to conduit 15. After the valve closes and the any remainingsample is locked in the post analysis conduit, the analyzer plungerretracts from the flexible diaphragm of the pump mean creating a partialvacuum in the sensor conduit. This forces the analysis fluid through thesmall hole in the tape gasket 31 and into a short transecting conduit inthe base, 13, 14. The analysis fluid is pulled further and the frontedge of the analysis fluid is oscillated across the surface of thesensor chip in order to shear the sample near the walls of the conduit.A conductivity sensor on the sensor chip is used to control thisprocess. The efficiency of the process is monitored using theamperometric sensors through the removal of unbound enzyme-antibodyconjugate which enhances the oxidation current measured at the electrodewhen the enzyme substrate, 4-aminophenyl phosphate is also present. Theamperometric electrodes are polarized to 0.06 V versus the silverchloride reference-ground electrode. In this embodiment, the fluid iscomposed of a 0.1 M carbonate or diethanolamine buffer, at pH 9.8, with1 mM MgCl₂, 1.0 M NaCl, 10 mM 4-aminophenylphosphate, and 10 μM NaI. Theefficiency of the wash is optimally further enhanced by introductioninto the fluid of one or more segments that segment the fluid within theconduit as previously described. The air segment may be introduced byeither active or passive means. Referring now to FIG. 14, there isillustrated the construction of a specific means for passivelyintroducing an air segment into said fluid. Within the base of theimmunosensor is recess 140 comprising a tapered portion 141 and acylindrical portion that are connected. The tapered portion is in fluidconnection with a hole 142 of similar diameter in the tape gasket (FIG.3) that separates the base (FIG. 4) and cover (FIGS. 1 and 2) of theassembled immunosensor cartridge. The recess contains an absorbentmaterial that, upon contact with fluid, withdraws a small quantity offluid from a conduit thereby passively introducing an air segment intothe conduit. The volume of the recess and the amount and type ofmaterial within it may be adjusted to control the size of the airsegment introduced. Specific materials include, but are not limited to,glass filter, a laminate comprising a 3 micron Versapor filter bonded bysucrose to a 60% viscose chiffon layer.

Fluid is forcibly moved towards sensor chip by the partial vacuumgenerated by reducing the mechanical pressure exerted upon paddle 6,causing the “T” region of the sensor channel in the vicinity of thetransecting conduit to fill with analysis fluid. The T region of thesensor channel optionally has a higher channel height resulting ameniscus with a smaller radius of curvature. Further away from the Tregion towards the post-analytical conduit, the conduit height isoptionally smaller. The analysis fluid passively flows from the T regiontowards this low conduit height region washing the conduit walls. Thispassive leak allows further effective washing of the T region using aminimal volume of fluid.

In this simple embodiment, the fluid located within the second conduitcontains a substrate for the enzyme. In other embodiments, amendment ofthe fluid using dried substrate within the second conduit may be used.

Following the positioning of a final segment of fluid over the sensor,measurement of the sensor response is recorded and the concentration ofanalyte determined as described for Example 2. Specifically, at leastone sensor reading of a sample is made by rapidly placing over thesensor a fresh portion of fluid containing a substrate for the enzyme.Rapid displacement both rinses away product previously formed, andprovides now substrate to the electrode. Repetitive signals are averagedto produce a measurement of higher precision, and also to obtain abetter statistical average of the baseline, represented by the currentimmediately following replacement of the solution over the sensor.

EXAMPLE 4

Referring now to FIG. 15, there is shown a top view of an immunosensorcartridge. Cartridge 150 comprises a base and a top portion, preferablyconstructed of a plastic. The two portions are connected by a thin,adhesive gasket or thin pliable film. As in previous embodiments, theassembled cartridge comprises a sample chamber 151 into which a samplecontaining an analyte of interest is introduced via a sample inlet 152.A metered portion of the sample is delivered to the sensor chip 153, viathe sample conduit 154 (first conduit) as before by the combined actionof a capillary stop 152, preferably formed by a 0.012″ laser cut hole inthe gasket or film that connects the two portions of the cartridge, andan entry point 155 located at a predetermined point within the samplechamber whereby air introduced by the action of a pump means, such as apaddle pushing upon a sample diaphragm 156. After contacting the sensorto permit binding to occur, the sample is moved to vent 157, whichcontains a wicking material that absorbs the sample and thereby sealsthe vent closed to the further passage of liquid or air. The wickingmaterial is preferably a cotton fiber material, a cellulose material, orother hydrophilic material having pores. It is important in the presentapplication that the material is sufficiently absorbent (i.e., possessessufficient wicking speed) that the valve closes within a time periodthat is commensurate with the subsequent withdrawal of the samplediaphragm actuating means described below, so that sample is notsubsequently drawn back into the region of the sensor chip.

As in the specific embodiments, there is provided a wash conduit (secondconduit) 158, connected at one end to a vent 159 and at the other end tothe sample conduit at a point 160 of the sample conduit that is locatedbetween vent 157 and sensor chip 153. Upon insertion of the cartridgeinto a reading apparatus, a fluid is introduced into conduit 158.Preferably, the fluid is present initially within a foil pouch 161 thatis punctured by a pin when an actuating means applies pressure upon thepouch. There is also provided a short conduit 162 that connects thefluid to conduit 154 via a small opening in the gasket 163. A secondcapillary stop initially prevents the fluid from reaching capillary stop160, so that the fluid is retained within conduit 158.

After vent 157 has closed, the pump means is actuated, creating alowered pressure within conduit 154. Air vent 164, preferably comprisinga small flap cut in the gasket or a membrane that vibrates to provide anintermittent air stream, provides a means for air to enter conduit 158via a second vent 165. The second vent 165 preferably also containswicking material capable of closing the vent if wetted, which permitssubsequent depression of sample diaphragm 156 to close vent 165, ifrequired. Simultaneously with the actuation of sample diaphragm 156,fluid is drawn from conduit 158, through capillary stop 160, intoconduit 154. Because the flow of fluid is interrupted by air enteringvent 164, at least one air segment (a segment or stream of segments) isintroduced.

Further withdrawal of sample diaphragm 156 draws the liquid containingat least one air segment back across the sensing surface of sensor chip153. The presence of air-liquid boundaries within the liquid enhancesthe rinsing of the sensor chip surface to remove remaining sample.Preferably, the movement of the sample diaphragm 156 is controlled inconjunction with signals received from the conductivity electrodeshoused within the sensor chip adjacent to the analyte sensors. In thisway, the presence of liquid over the sensor is detected, and multiplereadings can be performed by movement of the fluid in discrete steps.

It is advantageous in this embodiment to perform analyte measurementswhen only a thin film of fluid coats the sensors, ground chip 165, and acontiguous portion of the wall of conduit 154 between the sensors andground electrode. A suitable film is obtained by withdrawing fluid byoperation of the sample diaphragm 156, until the conductimetric sensorlocated next to the sensor indicates that bulk fluid is no longerpresent in that region of conduit 154. It has been found thatmeasurement can be performed at very low (nA) currents, the potentialdrop that results from increased resistance of a thin film betweenground chip and sensor chip (compared to bulk fluid), is notsignificant.

The ground chip 165 is preferably silver/silver chloride. It isadvantageous, to avoid air segments, which easily form upon therelatively hydrophobic silver chloride surface, to pattern the groundchip as small regions of silver/silver chloride interspersed with morehydrophilic regions, such as a surface of silicon dioxide. Thus, apreferred ground electrode configuration comprises an array ofsilver/silver chloride squares densely arranged and interspersed withsilicon dioxide. There is a further advantage in the avoidance ofunintentional segments if the regions of silver/silver chloride aresomewhat recessed.

Referring now to FIG. 16, there is shown a schematic view of thefluidics of the preferred embodiment of an immunosensor cartridge.Regions R1-R7 represent specific regions of the conduits associated withspecific operational functions. Thus R1 represents the sample chamber;R2 the sample conduit whereby a metered portion of the sample istransferred to the capture region, and in which the sample is optionallyamended with a substance coated upon the walls of the conduit; R3represents the capture region, which houses the conductimetric andanalyte sensors; R4 and R5 represent portions of the first conduit thatare optionally used for further amendment of fluids with substancescoated onto the conduit wall, whereby more complex assay schemes areachieved; R6 represents the portion of the second conduit into whichfluid is introduced upon insertion of the cartridge into a readingapparatus; R7 comprises a portion of the conduit located betweencapillary stops 160 and 166, in which further amendment can occur; andR8 represents the portion of conduit 154 located between point 160 andvent 157, and which can further be used to amend liquids containedwithin.

EXAMPLE 5 Coordination of Fluidics and Analyte Measurements

In the analysis sequence, a user places a sample into the cartridge,places the cartridge into the analyzer and in 1 to 20 minutes, aquantitative measurement of one or more analytes is performed. Herein isa non-limiting example of a sequence of events that occur during theanalysis:

1) A 25 to 50 uL sample is introduced in the sample inlet 167 and fillsto a capillary stop 151 formed by a 0.012″ laser cut hole in theadhesive tape holding the cover and base components together. The userrotates a latex rubber disk mounted on a snap flap to close the sampleinlet 167 and places the cartridge into the analyzer.

2) The analyzer makes contact with the cartridge, and a motor drivenplunger presses onto the foil pouch 161 forcing the wash/analysis fluidout into a central conduit 158.

3) A separate motor driven plunger contacts the sample diaphragm 156pushing a measured segment of the sample along the sample conduit (fromreagent region R1 to R2). The sample is detected at the sensor chip 153via the conductivity sensors. The sensor chip is located in captureregion R3.

4) The sample is oscillated by means of the sample diaphragm 156 betweenR2 and R5 in a predetermined and controlled fashion for a controlledtime to promote binding to the sensor.

5) The sample is pushed towards the waste region of the cartridge (R8)and comes in contact with a passive pump 157 in the form of a celluloseor similar absorbent wick. The action of wetting this wick seals thewick to air flow thus eliminating its ability to vent excess pressuregenerated by the sample diaphragm 156. The active vent becomes the“controlled air vent” of FIG. 16.

6) Rapid evacuation of the sample conduit (effected by withdrawing themotor driven plunger from the sample diaphragm 156) forces a mixture ofair (from the vent) and wash/analysis fluid from the second conduit tomove into the inlet located between R5 and R4 in FIG. 16. By repeatingthe rapid evacuation of the sample conduit, a series of air separatedfluid segments are generated which are pulled across the sensor chiptowards the sample inlet (from R4 to R3 to R2 and R1). This washes thesensor free of excess reagents and wets the sensor with reagentsappropriate for the analysis. The wash/analysis fluid which originatesin the foil pouch can be further amended by addition of reagents in R7and R6 within the central wash/analysis fluid conduit.

7) The wash/analysis fluid segment is drawn at a slower speed towardsthe sample inlet to yield a sensor chip which contains only a thin layerof the analysis fluid. The electrochemical analysis is performed at thispoint. The preferred method of analysis is amperometry but potentiometryor impedance detection is also used.

8) And the mechanism retracts allowing the cartridge to be removed fromthe analyzer.

Referring now to FIG. 17, there is illustrated an electrical signal 170representing the position of the electric motor actuating the samplediaphragm 156, the response 171 of the conductimetric electrode, and theelectrochemical response 172 of a amperometric immunosensor. In the timeperiod prior to 40 seconds after initiation of the immunoassay 173, themotor depresses the diaphragm, which pushes the sample into the captureregion and over the conductimetric sensor. Thus, after about 10 seconds,the conductivity rises to a steady value representative of samplefilling the portion of the conduit containing the conductimetric sensor.During this period the valve is sealed by contact with the sample.Between 40 seconds and about 63 seconds, the motor position is steppedback in increments 174, creating a periodic fluctuation in pressure,which draws an air-segmented portion of wash fluid over the sensor.During this period, fluctuations 175 in the immunoassay sensor are seen.At 177, the conductimetric response indicates that the wash fluid, whichcontains substrate, covers the conductimetric sensor. As the fluid isdrawn slowly over the sensor, a potential is applied (in this example,every five seconds, for 2.5 second periods) to the sensor, resulting inresponse 176, which indicates the presence of analyte bound to thesensor.

The invention described and disclosed herein has numerous benefits andadvantages compared to previous devices. These benefits and advantagesinclude, but are not limited to ease of use, the automation of most ifnot all steps of the analysis, which eliminates user included error inthe analysis.

EXAMPLE 6

In this example the amount of each component printed in the sampleholding chamber in the base (coating) is shown. The components are BSA,glycine, methoxypolyethylene glycol, sucrose and bromophenol blue (usedfor quality control—may be viewed by some as a helpful “target”). Thesample holding chamber in the base has to be corona treated in order toprint. The base cocktail is very dilute and won't spread without acorona treatment). The cover is not required to be corona treatedalthough it may be so treated in order to simplify operations. In apreferred embodiment there is no special treatment for the cover and notreatment around the orifice.

By way of example, the print cocktail may be made as follows. An aqueoussolution of bromophenol blue is prepared (0.05 g in 10 g of deionizedwater). A reagent mixture is prepared by dissolving BSA (0.42 g),glycine (2.54 g), MePEG (0.39 g) and sucrose (1.4 g) in 250 mL ofdeionized water. The print cocktail used to print into the cartridgecomponents constitutes reagent mixture (1.2 g), bromophenol bluesolution (0.23 g) mixed with deionized water (53.4 g).

PT Base Cocktail Bromophenol Blue Solution

BBlue 0.05 g Deionized water (DIW)   10 g  0.005 g/g

PT Matrix g g/g BSA 0.4288 0.001715 Glycine 2.538 0.010152 MePEG 0.39380.001575 Sucrose 1.4 0.0056 Total Volume 250

PT Base Print Cocktail: (g)

DIW 53.4 PT matrix 1.2 BBlue Solution 0.231 Total 54.831

base cocktail print 19ul (g) g/g stock g per component g/g g printed ugprinted PT Matrix-BSA 1.2 0.001715 0.00205824 3.75379E−05 7.1322E−070.71321989 PT Matrix-Glycine 1.2 0.010152 0.0121824 0.0002221814.2214E−06 4.22143678 PT Matrix-MePEG 1.2 0.001575 0.001890243.44739E−05  6.55E−07 0.65500465 PT Matrix-Sucrose 1.2 0.0056 0.006720.000122558 2.3286E−06 2.32860973 BBlue Solution 0.231 0.005 0.0011552.10647E−05 4.0023E−07 0.4002298

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

1-68. (canceled)
 69. An amperometric immunosensor, comprising: an electrochemical sensing surface for a blood sample having a porous polyvinyl alcohol layer patterned to cover at least a portion of the surface such that said layer attenuates background current from a blood sample to at least half the background current obtained in the absence of the layer.
 70. The immunosensor of claim 69, wherein an antibody is attached to said porous polyvinyl alcohol layer on latex particles.
 71. The immunosensor of claim 69, wherein the polyvinyl alcohol layer is patterned with a stilbizonium crosslinking agent and has a thickness 0.1 to 10 μm.
 72. The immunosensor of claim 69, wherein the polyvinyl alcohol layer is patterned with a stilbizonium crosslinking agent and has a thickness of about 0.6 μm.
 73. The immunosensor of claim 69, wherein the polyvinyl alcohol layer is patterned with a stilbizonium crosslinking agent and has a thickness of about 0.6 μm. 